Enhanced electrochemical detection using nanoparticles and precipitation

ABSTRACT

The invention described herein relates generally to methods, sensors, devices and kits for electrochemical detection of a target analyte in a sample. In certain aspects, the methods, sensors, devices and kits described herein can be used to detect low concentrations of at least one target analyte using small sample volumes. In some embodiments, methods, sensors and kits for detecting a microbe, microbe fragment or released endotoxin in a test sample, including bodily fluids such as blood and tissues of a subject, food, water, and environmental surfaces, are also provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 15/749,976, filed Feb. 2, 2018, which is a 35U.S.C. § 371 National Phase Entry of International Patent ApplicationNo. PCT/US2016/045369 filed Aug. 3, 2016 which claims benefit under 35U.S.C. § 119(e) of the U.S. Provisional Application No. 62/200,454,filed Aug. 3, 2015 contents of each of which are incorporated herein byreference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Contract No.N66001-11-1-4180 awarded by the Space and Warfare Systems Command. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 29, 2016, isnamed 002806-084981-PCT_SL.txt and is 18,380 bytes in size.

TECHNICAL FIELD

The invention described herein relates generally to methods, sensors,devices and kits for electrochemical detection of a target analyte in asample. In certain aspects, the methods, sensors, devices and kitsdescribed herein can be used to detect low concentrations of at leastone target analyte using small sample volumes. In some embodiments,methods, sensors and kits for detecting a microbe, microbe fragment orreleased endotoxin in a test sample, including bodily fluids such asblood and tissues of a subject, food, water, and environmental surfaces,are also provided herein.

BACKGROUND

Current immunoassays such as enzyme linked immunosorbent assay (ELISA)performed in 96-well microtiter plate requires a minimum sample volumeof 50 μL per well, i.e. 150 μL to perform the test in triplicate. Inaddition, the sample can only be tested for a single chemical orbiochemical (e.g. protein, toxin, drug) in a given well. Commercialalternatives for the multiplexed detection of several biochemicals exist(e.g. Luminex (Life Technologies Corp.) but are expensive, requireextensive preparative steps and relatively large sample volumes. Thereis a need for more sensitive, miniaturised and faster multiplexedassays. In addition, the immunoassay format limits the spatialresolution over the enzymatic reaction, i.e. the entire well turns“positive”. This makes difficult to address multiples analytes in asingle sample. Sample volumes become problematic. Colorimetricmicroarray-in-wells have been developed but lack sensitivity, andrequire expensive instrumentation (e.g. optics, laser scanner, etc.).

Electrochemical sensor platforms have been previously reported [seee.g., Wan, Y., et al., Development of electrochemical immunosensorstowards point of care diagnostics. Biosensors and Bioelectronics, 2013.47(0): p. 1-11. Ley, C., et al., An electrochemical microtiter plate forparallel spectroelectrochemical measurements. Electrochimica Acta, 2013.89(0): p. 98-105. Piermarini, S., et al., Electrochemical immunosensorarray using a 96-well screen-printed microplate for aflatoxin B1detection. Biosensors and Bioelectronics, 2007. 22(7): p. 1434-1440.Tang, T.-C., A. Deng, and H.-J. Huang, Immunoassay with a MicrotiterPlate Incorporated Multichannel Electrochemical Detection System.Analytical Chemistry, 2002. 74(11): p. 2617-2621. Castillo, J., et al.,Glutamate detection from nerve cells using a planar electrodes arrayintegrated in a microtiter plate. Biosensors and Bioelectronics, 2005.20(10): p. 2116-2119]. However, the existing electrochemical sensorplatforms are singleplex, i.e. only one electrode per well, dedicated tothe measurement of only one biochemical. The disposable electrode arraysare comparatively costly, as high-end electrodes arrays are ideallyphotolithographically microfabricated under clean room environment. Inaddition, diffusion of the oxidized substrate to neighboring electrodescan cause severe background current and lead to a number of errors inthe interpretation of the results.

The sensitive detection of pathogens/pathogen fragments/endotoxins witha sensitivity equal to 1 CFU/mL (CFU=colony forming unit) is achallenging task. Most systems rely on large equipment and tediousanalytical procedures. The ability to detect pathogens at those levelswould enable the development of a companion diagnostic able to provide arapid answer on the level of contamination present in a sample.Accordingly, there is a need to develop a method or approach that issensitive enough to detect low concentrations of analyte and is alsoversatile enough to detect multiple analytes simultaneously.

SUMMARY OF THE INVENTION

Certain aspects of the present invention described herein are, at leastin part, directed to a method for detecting a target analyte in asample, comprising:

-   -   (a) introducing a sample comprising a target analyte into an        electrochemical sensor comprising a fluid-contact surface and an        analyte-specific electrode immobilized on at least a portion of        the fluid-contact surface, wherein the analyte-specific        electrode is functionalized with a capture probe for specific        binding with the target analyte;    -   (b) allowing the target analyte to bind with the capture probe        on the analyte-specific electrode, thereby forming a complex        comprising the target analyte and the capture probe on a surface        of the analyte-specific electrode;    -   (c) labeling the complex with a label probe, wherein the label        probe binds specifically with the target analyte and the label        probe is conjugated with at least one reporter enzyme;    -   (d) introducing an electroactive mediator precipitating        composition into the electrochemical sensor, wherein a reaction        of the electroactive mediator precipitating composition with the        at least one reporter enzyme conjugated with the label probe        forms an electroactive precipitate locally adsorbed at the        surface of the analyte-specific electrode;    -   (e) applying a voltage to the electrochemical sensor, wherein        the voltage corresponds to the standard redox potential of the        electroactive precipitate; and    -   (f) measuring a current generated from the analyte-specific        electrode of the electrochemical sensor to detect the target        analyte;    -   wherein the target analyte is not a nucleic acid.

In some embodiments, the method further comprises prior to step (a):

-   -   i. mixing a sample comprising the target analyte with a        plurality of nanoparticles, wherein at least one nanoparticle of        said plurality of nanoparticles is functionalized with a capture        probe for specific binding with the target analyte; and    -   ii. allowing the target analyte to bind with the capture probe        on said at least one nanoparticle.

In some embodiments, the electrochemical sensor comprises a plurality ofanalyte-specific electrodes immobilized on at least a portion of thefluid-contact surface, wherein each analyte-specific electrode in saidplurality of analyte-specific electrodes is functionalized with acapture probe for specific binding with a specific target analyte. Insome embodiments, at least two of the analyte-specific electrodes areadapted to detect different target analytes. In some embodiments, atleast two different target analytes in the sample are detected.

In some embodiments, the target analyte is selected from the groupconsisting of a protein, a peptide, a polypeptide, a peptidomimetic, anantibody, an antibody fragment, an amino acid, a peptide aptamer, apeptidoglycan, a cell, microbial matter, a carbohydrate, an antigen, alipid, a steroid, a hormone, a lipopolysaccharide, an endotoxin, a drug,a lipid-binding molecule, a cofactor, a small molecule, a toxin, and anycombination thereof. In some embodiments, the protein is a glycoprotein.Exemplary microbial matter include, but are not limited to, bacteria,viruses, protozoa, fungi, yeast, microbes, parasites, any fragmentsthereof, and any combination thereof. Exemplary carbohydrates include,but are not limited to, mannose, mannan, N-acetyl glucosamine, fucose, amonosaccharide, a disaccharide, a trisaccharide, a polysaccharide, andany combination thereof.

In some embodiments comprising a nanoparticle, the nanoparticle may be amagnetic nanoparticle, a gold nanoparticle, a silver nanoparticle, asemiconductor nanoparticle, or a polymeric nanoparticle. In someembodiments, at least two of the nanoparticles are functionalized withcapture probes for specific binding with at least two different targetanalytes.

Some embodiments of the method further comprise, prior to the step ofapplying the voltage to the electrochemical sensor, washing theelectrochemical sensor to remove any electroactive mediatorprecipitating composition or electroactive precipitate that is notadsorbed at the analyte-specific electrode surface.

In some embodiments, the electrochemical sensor comprises one or moremicrofluidic flow cells. In some embodiments, the electrochemical sensorcomprises one or more open wells. Some embodiments comprise both one ormore microfluidic flow cells and one or more open wells.

In some embodiments, the analyte-specific electrode is a planar or3-dimensional electrode. In some embodiments, the analyte-specificelectrode comprises gold, silver, copper, platinum, aluminum, stainlesssteel, tungsten, indium tin oxide, titanium, lead, nickel, palladium,silicon, polyimide, parylene, benzocyclobutene, carbon, graphite, or anycombination thereof. In some embodiments, the fluid-contact surfacefurther comprises a counter electrode, a reference electrode, a positivecontrol electrode, a negative control electrode, or any combinationthereof immobilized thereon.

In some embodiments, the voltage applied to the electrochemical sensorcorresponds to an electrochemical oxidation or reduction potential, orcombination thereof, of the electroactive mediator in a fully orpartially oxidized state. In some embodiments, the generated currentcorresponds to a reduction or oxidation current derived from reductionor oxidation of the fully or partially oxidized electroactive mediator.An exemplary voltage window includes, but is not limited to, about −0.2Vas reduction potential to +0.2V as oxidation potential versus areference electrode.

In some embodiments, the fluid-contact surface is a non-electricallyconductive surface. Exemplary non-electrically conductive surfacesinclude, but are not limited to, plastic, poly(carbonate) (PC),poly(methyl methacrylate) (PMMA), cyclic olefin polymers (COP), cyclicolefin copolymers (COC), silicon nitride, parylene, kapton,styrene-ethylene-butylene-styrene (SEBS), poly-dimethysiloxane (PDMS),polyimide, silicon dioxide, and any combination thereof.

In some embodiments, the capture probe and the label probe areindependently selected from the group consisting of an antibody, anantibody fragment, a carbohydrate-binding protein, a peptide, apolypeptide, an aptamer, a cell-binding molecule, a lipid-bindingmolecule, and any combination thereof. In some embodiments, the targetanalyte comprises a microbe, and the capture probe and label probecomprise a carbohydrate binding protein, wherein the carbohydratebinding protein comprises a carbohydrate recognition domain ofmannan-binding lectin (MBL). In some embodiments, the carbohydraterecognition domain of MBL is conjugated to an Fc portion of animmunoglobin.

In some embodiments, at least one reporter enzyme is conjugated to thelabel probe before the label probe binds to the target analyte complex.In other embodiments, at least one reporter enzyme is conjugated to thelabel probe after the label probe binds to the target analyte complex.In some embodiments, the label probe is functionalized with biotin andsaid at least one reporter enzyme is conjugated to streptavidin. In someembodiments, the label probe first binds to the target analyte complex,and then the streptavidin conjugated to said at least one reporterenzyme binds to the biotin functionalized label probe.

In some embodiments, at least one reporter enzyme comprises horseradishperoxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOx),tyrosinase, urease, a DNAzyme, an aptazyme, or any combination thereof.In some embodiments, at least one reporter enzyme comprises HRP.

In some embodiments, the electroactive mediator precipitatingcomposition comprises a reporter enzyme substrate and an electroactivemediator. Exemplary reporter enzyme substrates include, but are notlimited to, hydrogen peroxide, carbamide peroxide, nucleotides,oligonucleotides, RNA, DNA, phosphorylated peptides, phosphorylatedproteins, phosphorylated small molecules, glucose, phenols, tyrosine,dopamine, catechol, urea, and any combination thereof. In someembodiments, the reporter enzyme substrate is hydrogen peroxide.Exemplary electroactive mediators include, but are not limited to,3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride(OPD), 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS),p-Nitrophenyl Phosphate (PNPP), 3,3′-diaminobenzidine (DAB),4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP),nitro blue tetrazolium (NBT), methylene blue, hydroquinone, ferrocenederivatives, and any combination thereof. In some embodiments, theelectroactive mediator is TMB. In some embodiments, the electroactivemediator precipitating composition further comprises a precipitatingagent. Exemplary precipitating agents include, but are not limited to, awater-soluble polymer, a pyrrolidinone polymer, a polyaniline, apolypyrrole, a polythiophene, alginic acid, methyl vinyl ether/maleicanhydride copolymer, dextran sulfate, carrageenan, and any combinationthereof. In some embodiments, the precipitating agent is a pyrrolidinonepolymer.

Certain aspects of the present invention described herein are, at leastin part, directed to a kit for electrochemical multiplex detection of aplurality of target analytes in a sample comprising:

-   -   (a) an electrochemical sensor comprising a fluid-contact surface        and a plurality of analyte-specific electrodes immobilized on at        least a portion of the fluid-contact surface, wherein the        analyte-specific electrodes are each functionalized with a        capture probe for binding a specific target analyte;    -   (b) a plurality of label probes, wherein each label probe is for        binding a specific target analyte, and wherein each label probe        is conjugated to at least one reporter enzyme or is        functionalized to be conjugated to at least one reporter enzyme;        and    -   (c) an electroactive mediator precipitating composition        comprising a reporter enzyme substrate, an electroactive        mediator and a precipitating agent, wherein a reaction of the        reporter enzyme substrate and the electroactive mediator with        the reporter enzyme forms an electroactive precipitate locally        adsorbed at the surface of the analyte-specific electrodes;    -   wherein none of the target analytes are nucleic acids.

Some embodiments further comprise a plurality of nanoparticles, whereinat least one nanoparticle of said plurality of nanoparticles isfunctionalized with a capture probe for specific binding with a targetanalyte. Exemplary nanoparticles include, but are not limited to, amagnetic nanoparticle, a gold nanoparticle, a silver nanoparticle, asemiconductor nanoparticle, or a polymeric nanoparticle.

In some embodiments, the electrochemical sensor comprises one or moreopen wells. In some embodiments, the electrochemical sensor comprisesone or more microfluidic flow cells. Some embodiments comprise both, oneor more microfluidic flow cells and one or more open wells.

In some embodiments, the capture probe and the label probe areindependently selected from the group consisting of an antibody, anantibody fragment, a carbohydrate-binding protein, a peptide, apolypeptide, an aptamer, a cell-binding molecule, a lipid-bindingmolecule, and any combination thereof. In some embodiments, the targetanalyte comprises a microbe, and the capture probe and label probecomprise a carbohydrate binding protein, wherein the carbohydratebinding protein comprises a carbohydrate recognition domain ofmannan-binding lectin (MBL). In some embodiments, the carbohydraterecognition domain of MBL is conjugated to an Fc portion of animmunoglobin.

In some embodiments, the label probes are functionalized with biotin andthe reporter enzymes are conjugated to streptavidin, so that the labelprobes first bind to specific target analytes, and then the streptavidinconjugated to the reporter enzymes binds to the biotin functionalizedlabel probes. Exemplary reporter enzymes include, but are not limitedto, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucoseoxidase (GOx), tyrosinase, urease, a DNAzyme, a aptazyme, or anycombination thereof. In some embodiments, at least one reporter enzymecomprises HRP.

In some embodiments, the electroactive mediator precipitatingcomposition comprises a reporter enzyme substrate and an electroactivemediator. Exemplary reporter enzyme substrates include, but are notlimited to, hydrogen peroxide, carbamide peroxide, nucleotides,oligonucleotides, RNA, DNA, phosphorylated peptides, phosphorylatedproteins, phosphorylated small molecules, glucose, phenols, tyrosine,dopamine, catechol, urea, and any combination thereof. In someembodiments, the reporter enzyme substrate is hydrogen peroxide.Exemplary electroactive mediators include, but are not limited to,3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride(OPD), 2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS),p-Nitrophenyl Phosphate (PNPP), 3,3′-diaminobenzidine (DAB),4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP),nitro blue tetrazolium (NBT), methylene blue, hydroquinone, ferrocenederivatives, and any combination thereof. In some embodiments, theelectroactive mediator is TMB. In some embodiments, the electroactivemediator precipitating composition further comprises a precipitatingagent. Exemplary precipitating agents include, but are not limited to, awater-soluble polymer, a pyrrolidinone polymer, a polyaniline, apolypyrrole, a polythiophene, alginic acid, methyl vinyl ether/maleicanhydride copolymer, dextran sulfate, carrageenan, and any combinationthereof. In some embodiments, the precipitating agent is a pyrrolidinonepolymer.

Certain aspects of the present invention described herein are, at leastin part, directed to an electrochemical sensor comprising:

-   -   (a) a fluid-contact surface and a plurality of analyte-specific        electrodes immobilized on at least a portion of the        fluid-contact surface, wherein the analyte-specific electrodes        are each functionalized with a capture probe for binding a        specific target analyte;    -   (b) a plurality of different nanoparticle-bound target analytes        bound to the corresponding capture probes of the        analyte-specific electrodes; and    -   (c) an electroactive precipitate locally adsorbed at the        surfaces of at least some of the analyte-specific electrodes,        wherein the electroactive precipitate is formed from a reaction        of an electroactive mediator precipitating composition        comprising a reporter enzyme substrate, an electroactive        mediator and a precipitating agent, with a reporter enzyme        coupled to the nanoparticle-bound target analytes;        wherein none of the target analytes are nucleic acids.

In some embodiments, the reporter enzyme is coupled to thenanoparticle-bound target analytes by specific binding of a label probeto the corresponding nanoparticle-bound target analytes, wherein thelabel probe is conjugated to the reporter enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an embodiment of a method forelectrochemical detection of at least one target analyte in a sample.

FIGS. 2A and 2B show the effect of sample pretreatment with the additionof nanoparticles on sensor sensitivity.

FIG. 3A is a schematic representation of a 32-electrode array arrangedin a single 6 mm diameter well, according to one embodiment. (Outer darkgrey ring: counter electrode; Green electrodes: antigen specificelectrodes; Red and dark green electrodes: positive and negativecontrols respectively; Light grey central electrode: referenceelectrode).

FIG. 3B is a calibration curve obtained for the electrochemicaldetection of the inflammation protein interleukin-6 (IL-6) in theconcentration range of 2 fg/mL-20 pg/mL on a gold electrode. Currentmeasured is proportional to the amount of enzyme bound to the electrode,and therefore to the antigen concentration.

FIGS. 4A and 4B each show sensor chips according to embodiments of theinvention and consisting of 64 sensing electrodes, individuallyaddressable. Each sensing electrode is functionalized with a givencapture probe (e.g. antibody, carbohydrate-binding protein, syntheticbinding element, etc.). FIG. 4A shows an open well embodiment. FIG. 4Bshows a flow cell embodiment. In these embodiments, the open wells ormicrofluidic cells are glued on top of the electrode array and used toconfine samples and introduce various reagents and washing buffers toperform the assay steps. An antibody for IL-6 was immobilized usingstandard coupling chemistry.

FIG. 5 shows a comparison between labeling with single HRP vs. poly-HRPconjugated streptavidin.

FIG. 6 shows a full cross-reactivity study of anti-IL-6 modified sensorsexposed to various individual chemokines and mixtures.

FIG. 7 shows electrochemical detection of mannan in TBS-Tween Ca²⁺buffer using a biotin-FcMBL labeling approach.

FIG. 8 shows electrochemical detection of mannan in TBS-Tween Ca²⁺buffer using a HRP-RhMBL labeling approach.

FIG. 9A is a photograph showing a 64-electrode electrochemical sensorchip according to an embodiment of the invention.

FIG. 9B is an enlarged photograph of the 64-electrode electrochemicalsensor chip shown in FIG. 9A and depicts the working electrode,reference electrode and counter electrode.

FIG. 10 is a schematic representation of a multiplex assay principle.From left to right, A represents a bare sensor; B represents the sensormodified with antibody; C represents the sensor with target proteinbound to antibody when a sample is injected; D represents the sensorwhen HRP label detection antibody binds to antibody-protein complex; Erepresents the sensor when the introduced enzyme substrate reacts withtethered enzyme; and F represents the sensor when enzymatic productprecipitates out of solution onto the sensor.

FIG. 11 is a graph showing a typical recorded signal. The potential isscanned from negative to positive, and the current produced due to thepresence of the enzymatic product is measured. The signal can bereported as max peak current (I) and/or integrated and reported ascharge (C). I or C is proportional to quantity of enzyme bound to theelectrode, therefore to the detection antibody, and therefore to theamount of bound protein.

FIGS. 12A and 12B are calibration curves for the simultaneouselectrochemical detection of the two inflammatory markers IP-10 and IL-6in culture media.

FIGS. 13A and 13B are bar graphs showing reproducibility across singlearray 5.5%, 100 pg/mL, n=18 for protein sensors and n=6 for controls(FIG. 13A), and cross reactivity and simultaneous protein detection(FIG. 13B).

FIG. 14A is a bargraph showing side-to-side comparison of the calculatedconcentration measured using traditional ELISA kits (black bars)necessitating 1 kit for each protein and simultaneously measuring on theelectrochemical plaftorm (light grey bars). (NI: non infected chip; RV:Rhinovirus infected chip).

FIG. 14B shows a correlation graph for all concentration presented inFIG. 14A. The correlation was excellent with an r² value of 0.9947.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of various aspects described herein relate to methods,compositions, devices and kits for detecting at least one targetanalyte. Some embodiments provided herein relates to methods ofdetecting at least one target analyte, including, e.g., at least 2, 3,4, 5, 6, 7, 8 target analytes or more. In some embodiments, a highlysensitive electrochemical detection method is disclosed for detectingand/or quantifying target analytes (e.g., proteins, carbohydrates,glycoproteins, cells, pathogens, pathogens fragments, releasedendotoxins, etc.). Advantageously, some embodiments of the disclosedmethod increase the sensitivity of detection by contacting a samplecomprising a target analyte with nanoparticles coated with a captureprobe specific for the target analyte prior to introducing the sample toan electrochemical sensor. In some embodiments, the combination of (i)localized electrochemical detection using an electroactive precipitateand (ii) sample pretreatment using nanoparticles coated with a captureprobe, enables the highly sensitive detection of the target analyte andachieves multiplex target detection from a single sample, i.e., severaltarget analytes can be detected in a single assay or well with minimalor no chemical cross-talk between electrodes due to the localizedadsorption of electroactive precipitate.

In some embodiments, two or more target analytes can be detectedsimultaneously using an electrochemical sensor having one or more wells.As used herein, the term “detect” includes identifying the presence orabsence of one or more target analytes, and can also include quantifyingthe amount and/or concentration of one or more target analytes in thesample. In some embodiments, each well of the electrochemical sensorcomprises an inner bottom surface on which one or more analyte specificelectrodes is immobilized. In some embodiments, the wells are open cellscomprising open tops, enclosed sides and bottom, and one or moreanalyte-specific electrodes immobilized on the inner fluid-contactsurface of the wells. In some embodiments, the electrochemical sensorcomprises 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 32, 48, 64, 96 or moreopen wells. FIG. 4A depicts an open cell embodiment having 8 open wells,and 8 electrodes in each well. Another embodiment is in the form of a96-well microtiter plate. In some embodiments, the wells aremicrofluidic flow cells comprising an enclosed top, sides and bottom,wherein the top of each flow cell includes a fluid inlet and a fluidoutlet, and comprising one or more analyte-specific electrodesimmobilized on the inner fluid-contact surface of the wells. In someembodiments, the electrochemical sensor comprises 1, 2, 3, 4, 5, 6, 8,10, 12, 16, 24, 32, 48, 64, 96 or more microfluidic flow cells. FIG. 4Bdepicts a flow cell embodiment having 8 enclosed wells, and 8 electrodesin each well. Another embodiment is in the form of a 96-well microtiterplate, wherein each well comprises an enclosed top having a fluid inletand a fluid outlet. In some embodiments, the electrochemical sensorcomprises both one or more open cells and one or more flow cells. Eachwell contains an array of analyte-specific electrodes (e.g., 32 goldelectrodes) that can be individually modified with capture probes tobind the corresponding target analyte (e.g., pathogen, protein,carbohydrate, toxin, drug, etc.) present in the collected sample. Insome embodiments, one sample is introduced into each well. Inembodiments having two or more wells, portions of the same sample can beintroduced into more than one well, or different samples can beintroduced into different wells. Thus, in embodiments having multiplewells, multiple samples can be simultaneously assayed.

In some embodiments, the electrochemical sensor comprises (i) theanalyte-specific electrodes on which the capture probes are immobilized;(ii) a contact pad, which connects the electrodes (e.g.,analyte-specific electrodes, control electrodes, reference electrodes,etc.) to a measuring unit (i.e., readout instrumentation) (see forexample the gold contact pads on the outer perimeter of the open celland flow cell embodiments shown in FIGS. 4A-4B); and (iii) a conductivetrack that links (i) to (ii) (see for example the gold leads connectingthe electrodes to the gold contact pads in the embodiments shown inFIGS. 4A-4B). In general, (iii) is not exposed to fluid samples, (iii)can be covered by a polymer layer (e.g., SU-8) or simply hidden from thefluid sample using microfluidics.

As used herein, an “electrode” is an electrical conductor used to makecontact with a nonmetallic part of a circuit (i.e., it emits or collectselectrons or electron “holes”). Electrodes can comprise any electricallyconducting or semi-conducting material. Non-limiting examples includegold, silver, copper, platinum, aluminum, stainless steel, tungsten,indium tin oxide, titanium, lead, nickel, silicon, polyimide, parylene,benzocyclobutene, carbon, graphite, or any combination thereof.Preferably, electrodes comprise gold. The use of inexpensive gold-coatedprinted circuit board (PCB) substrates as electrochemical sensorplatform has been reported [La Belle, J. T., et al., Label-FreeImpedimetric Detection of Glycan-Lectin Interactions. AnalyticalChemistry, 2007. 79(18): p. 6959-6964. Umek, R. M., et al., ElectronicDetection of Nucleic Acids: A Versatile Platform for MolecularDiagnostics. The Journal of Molecular Diagnostics, 2001. 3(2): p. 74-84.Lian, K., et al., Integrated microfluidic components on a printed wiringboard platform. Sensors and Actuators B: Chemical, 2009. 138(1): p.21-27]. Metal patterning techniques, such as standard PCB technology,offer a number of versatile fabrication options such as (i) track sizeand spacing less than 100 μm; (ii) high purity electrolytic gold platingseveral microns thick suitable for electrochemistry and surfacemodification chemistries; (iii) ease of small scale prototyping instandard laboratory settings; and (iv) large scale mass manufacturingcapabilities at a fraction of the cost of high-end microarrays. In someembodiments, electrodes as disclosed herein may be fabricated using PCBtechnology.

In some embodiments, the electrodes are mass fabricated ontonon-electrically conductive surfaces such as plastic substrates usinginexpensive standard technology such as printed circuit board (PCB)technology, roll-to-roll laser ablation or evaporation. Exemplarynon-electrically conductive surfaces include plastic, poly(carbonate)(PC), poly(methyl methacrylate) (PMMA), cyclic olefin polymers (COP) orcyclic olefin copolymers (COC), SU-8, parylene, silicon nitride, kapton,styrene-ethylene-butylene-styrene (SEBS), poly-dimethysiloxane (PDMS),polyimide, silicon dioxide, and any combination thereof.

In some embodiments, the electrode is a planar or a 3-dimensionalelectrode. As used herein, a planar electrode electrically interactswith an electroactive species or mediator on a 2-dimensional surface. Asused herein, a 3-dimensional electrode is an electrode displaying a veryhigh surface area per unit volume, caused by no planarity. Without beingbound by theory, this provides high turbulence at their interface withan electroactive species or mediator, enhancing the mass transferprocess of the electroactive species towards the electrode surface.These characteristics strongly improve the electrochemical reactionrate.

Types of electrodes include analyte-specific electrodes, positivecontrol electrodes, negative control electrodes, counter electrodes,reference electrodes, among other types. As used herein,“analyte-specific electrodes” are electrodes coated or otherwisefunctionalized with a capture probe for specific binding with a targetanalyte.

In some embodiments, the electrochemical sensor comprises 1, 2, 3, 4, 5,6, 8, 10, 12, 16, 24, 32, 48 or more electrodes. In one embodiment, eachwell of the electrochemical sensor comprises 32 gold electrodes, whichcan enable the simultaneous detection of 1, 2, 3, 4, 5, 6, 7, or 8different target analytes in triplicate, including positive and negativecontrols.

In some embodiments, the sensors consist of 300 μm diameter workingelectrodes that can be individually modified with receptor proteins(e.g. antibody, FcMBL) to capture their respective molecular targets. Areference electrode is used to control the potential or current appliedat the working electrode and the resulting current is measured betweenthe working and counter electrodes.

Generally, a single chip can be used to detect up to 64 proteinssimultaneously. However, in some embodiments, measurements are realizedin triplicate, including positive and negative controls. In someembodiments, negative controls consist of electrodes blocked with bovineserum albumin (BSA), for example, to limit non-specific adsorption.Negative controls allow monitoring background readings and eventuallycorrect the protein sensor. In some embodiments, positive controlelectrodes are modified with BSA-biotin which can bind streptavidin-HRP,the label used in the last step of the assay in some embodiments. Thiscan be used to confirm that all assay steps were realized successfullyand that the chips are connected properly. The readings from positivecontrols can be used to normalize the protein sensors readings andcompensate wells-to-wells variations.

In some embodiments, the target analyte can include a biological cellselected from the group consisting of living or dead cells (prokaryoticand eukaryotic, including mammalian), viruses, bacteria, fungi, yeast,protozoan, microbes, and parasites. The biological cell can be a normalcell or a diseased cell, e.g., a cancer cell. Mammalian cells include,without limitation; primate, human and a cell from any animal ofinterest, including without limitation; mouse, hamster, rabbit, dog,cat, domestic animals, such as equine, bovine, murine, ovine, canine,and feline. In some embodiments, the cells can be derived from a humansubject. In other embodiments, the cells are derived from a domesticatedanimal, e.g., a dog or a cat. Exemplary mammalian cells include, but arenot limited to, stem cells, cancer cells, progenitor cells, immunecells, blood cells, fetal cells, and any combinations thereof. The cellscan be derived from a wide variety of tissue types without limitationsuch as, hematopoietic, neural, mesenchymal, cutaneous, mucosal,stromal, muscle, spleen, reticuloendothelial, epithelial, endothelial,hepatic, kidney, gastrointestinal, pulmonary, cardiovascular, T-cells,and fetus. Stem cells, embryonic stem (ES) cells, ES-derived cells,induced pluripotent stem cells, and stem cell progenitors are alsoincluded, including without limitation, hematopoietic, neural, stromal,muscle, cardiovascular, hepatic, pulmonary, and gastrointestinal stemcells. Yeast cells may also be used as cells in some embodimentsdescribed herein. In some embodiments, the cells can be ex vivo orcultured cells, e.g. in vitro. For example, for ex vivo cells, cells canbe obtained from a subject, where the subject is healthy and/or affectedwith a disease. While cells can be obtained from a fluid sample, e.g., ablood sample, cells can also be obtained, as a non-limiting example, bybiopsy or other surgical means known to those skilled in the art.

In some embodiments, the target analyte refers to a rare cell or acellular component thereof. In some embodiments, the target analyte canrefer to a rare cell or a cellular component thereof derived from amammalian subject, including, without limitation, primate, human or anyanimal of interest such as mouse, hamster, rabbit, dog, cat, domesticanimals, such as equine, bovine, murine, ovine, canine, and feline. Insome embodiments, the rare cells can be derived from a human subject. Inother embodiments, the rare cells can be derived from a domesticatedanimal or a pet such as a cat or a dog. As used herein, the term “rarecells” is defined, in some embodiments, as cells that are not normallypresent in a fluid sample, e.g., a biological fluid sample, but can bepresent as an indicator of an abnormal condition, such as infectiousdisease, chronic disease, injury, proliferative diseases, or pregnancy.In some embodiments, the term “rare cells” as used herein refers tocells that can be normally present in biological specimens, but arepresent with a frequency several orders of magnitude (e.g., at leastabout 100-fold, at least about 1000-fold, at least about 10000-fold)less than other cells typically present in a normal biological specimen.In some embodiments, rare cells are found infrequently in circulatingblood, e.g., less than 100 cells (including less than 10 cells, lessthan 1 cell) per 10⁸ mononuclear cells in about 50 mL of peripheralblood. In some embodiments, a rare cell can be a normal cell or adiseased cell. Examples of rare cells include, but are not limited to,circulating tumor cells, progenitor cells, e.g., collected for bonemarrow transplantation, blood vessel-forming progenitor cells, stemcells, circulating fetal cells, e.g., in maternal peripheral blood forprenatal diagnosis, virally-infected cells, cell subsets collected andmanipulated for cell and gene therapy, and cell subpopulations purifiedfor subsequent gene expression or proteomic analysis, other cellsrelated to disease progression, and any combinations thereof.

As used herein, the term “a cellular component” in reference tocirculating tumor cells, stem cells, fetal cells and/or microbes isintended to include any component of a cell that can be at leastpartially isolated from the cell, e.g., upon lysis of the cell. Cellularcomponents can include, but are not limited to, organelles, such asnuclei, perinuclear compartments, nuclear membranes, mitochondria,chloroplasts, or cell membranes; polymers or molecular complexes, suchas lipids, polysaccharides, proteins (membrane, trans-membrane, orcytosolic); nucleic acids, viral particles, or ribosomes; or othermolecules, such as hormones, ions, and cofactors.

As used herein, the term “toxin” refers to a compound produced by anorganism which causes or initiates the development of a noxious,poisonous or deleterious effect in a host presented with the toxin. Suchdeleterious conditions may include fever, nausea, diarrhea, weight loss,neurologic disorders, renal disorders, hemorrhage, and the like. Toxinsinclude, but are not limited to, bacterial toxins, such as choleratoxin, heat-liable and heat-stable toxins of E. coli, toxins A and B ofClostridium difficile, aerolysins, and hemolysins; toxins produced byprotozoa, such as Giardia; toxins produced by fungi. Molecular toxinscan also include exotoxins, i.e., toxins secreted by an organism as anextracellular product, and enterotoxins, i.e., toxins present in the gutof an organism.

In some embodiments, the method described herein can be used to detectat least one of the following pathogens that causes diseases: Bartonellahenselae, Borrelia burgdorferi, Campylobacter jejuni,Campylobacterfetus, Chlamydia trachomatis, Chlamydia pneumoniae,Chylamydia psittaci, Simkania negevensis, Escherichia coli (e.g.,O157:H7 and K88), Ehrlichia chafeensis, Clostridium botulinum,Clostridium perfringens, Clostridium tetani, Enterococcus faecalis,Haemophilius influenzae, Haemophilius ducreyi, Coccidioides immitis,Bordetella pertussis, Coxiella burnetii, Ureaplasma urealyticum,Mycoplasma genitalium, Trichomatis vaginalis, Helicobacter pylori,Helicobacter hepaticus, Legionella pneumophila, Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium africanum,Mycobacterium leprae, Mycobacterium asiaticum, Mycobacterium avium,Mycobacterium celatum, Mycobacterium celonae, Mycobacterium fortuitum,Mycobacterium genavense, Mycobacterium haemophilum, Mycobacteriumintracellulare, Mycobacterium kansasii, Mycobacterium malmoense,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium simiae,Mycobacterium szulgai, Mycobacterium ulcerans, Mycobacterium xenopi,Corynebacterium diptheriae, Rhodococcus equi, Rickettsia aeschlimannii,Rickettsia africae, Rickettsia conorii, Arcanobacterium haemolyticum,Bacillus anthracis, Bacillus cereus, Lysteria monocytogenes, Yersiniapestis, Yersinia enterocolitica, Shigella dysenteriae, Neisseriameningitides, Neisseria gonorrhoeae, Streptococcus bovis, Streptococcushemolyticus, Streptococcus mutans, Streptococcus pyogenes, Streptococcuspneumoniae, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus pneumoniae, Staphylococcus saprophyticus, Vibriocholerae, Vibrio parahaemolyticus, Salmonella typhi, Salmonellaparatyphi, Salmonella enteritidis, Treponema pallidum, Human rhinovirus,Human coronavirus, Dengue virus, Filoviruses (e.g., Marburg and Ebolaviruses), Hantavirus, Rift Valley virus, Hepatitis B, C, and E, HumanImmunodeficiency Virus (e.g., HIV-1, HIV-2), HHV-8, Humanpapillomavirus, Herpes virus (e.g., HV-I and HV-II), Human T-celllymphotrophic viruses (e.g., HTLV-I and HTLV-II), Bovine leukemia virus,Influenza virus, Guanarito virus, Lassa virus, Measles virus, Rubellavirus, Mumps virus, Chickenpox (Varicella virus), Monkey pox, EpsteinBahr virus, Norwalk (and Norwalk-like) viruses, Rotavirus, ParvovirusB19, Hantaan virus, Sin Nombre virus, Venezuelan equine encephalitis,Sabia virus, West Nile virus, Yellow Fever virus, causative agents oftransmissible spongiform encephalopathies, Creutzfeldt-Jakob diseaseagent, variant Creutzfeldt-Jakob disease agent, Candida, Cryptcooccus,Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax,Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes,Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoonhellem, Encephalitozoon cuniculi, among other viruses, bacteria,archaea, protozoa, and fungi).

In some embodiments, the method described herein can be used to detectbacteria present in a biofilm. For example, Listeria monocytogenes canform biofilms on a variety of materials used in food processingequipment and other food and non-food contact surfaces (Blackman, J FoodProt 1996; 59:827-31; Frank, J Food Prot 1990; 53:550-4; Krysinski, JFood Prot 1992; 55:246-51; Ronner, J Food Prot 1993; 56:750-8). Biofilmscan be broadly defined as microbial cells attached to a surface, andwhich are embedded in a matrix of extracellular polymeric substancesproduced by the microorganisms. Biofilms are known to occur in manyenvironments and frequently lead to a wide diversity of undesirableeffects. For example, biofilms cause fouling of industrial equipmentsuch as heat exchangers, pipelines, and ship hulls, resulting in reducedheat transfer, energy loss, increased fluid frictional resistance, andaccelerated corrosion. Biofilm accumulation on teeth and gums, urinaryand intestinal tracts, and implanted medical devices such as cathetersand prostheses frequently lead to infections (Characklis W G. Biofilmprocesses. In: Characklis W G and Marshall K C eds. New York: John Wiley& Sons, 1990:195-231; Costerton et al., Annu Rev Microbiol 1995;49:711-45).

In some embodiments, the method described herein can be used to detect aplant pathogen. Plant fungi have caused major epidemics with hugesocietal impacts. Examples of plant fungi include, but are not limitedto, Phytophthora infestans, Crinipellis perniciosa, frosty pod(Moniliophthora roreri), oomycete Phytophthora capsici, Mycosphaerellafijiensis, Fusarium Ganoderma spp fungi and Phytophthora. An exemplaryplant bacterium includes Burkholderia cepacia. Exemplary plant virusesinclude, but are not limited to, soybean mosaic virus, bean pod mottlevirus, tobacco ring spot virus, barley yellow dwarf virus, wheat spindlestreak virus, soil born mosaic virus, wheat streak virus in maize, maizedwarf mosaic virus, maize chlorotic dwarf virus, cucumber mosaic virus,tobacco mosaic virus, alfalfa mosaic virus, potato virus X, potato virusY, potato leaf roll virus and tomato golden mosaic virus.

In yet other embodiments, the method described herein can be used todetect bioterror agents (e.g., B. Anthracis, and smallpox).

As used herein, the terms “capture probe” and “label probe” are used todescribe an agent configured to detect and/or capture at least onetarget analyte as described herein. That is, the capture probe and labelprobe specifically bind to the target analyte to be detected. Thecapture probe can be present in any form, including but not limited to atarget-binding molecule, and/or a target-binding substrate (e.g., atarget-binding molecule conjugated to a solid substrate or a solidsupporting structure such as an analyte specific electrode or ananoparticle). As the present invention is directed to electrochemicaldetection of target analytes which are not nucleic acids (e.g., not DNA,not RNA, not oligonucleotides, etc.), the terms “capture probe” and“label probe” as used herein do not include nucleic acids. In someembodiments, the capture probe and/or label probe can comprise atarget-binding molecule selected from the group consisting of peptides,polypeptides, proteins, peptidomimetics, antibodies, antibody fragments(e.g., antigen binding fragments of antibodies), carbohydrate-bindingproteins (e.g., lectins, glycoproteins, glycoprotein-binding molecules),amino acids, carbohydrates (e.g., mono-, di-, tri- andpoly-saccharides), lipids, steroids, hormones, lipid-binding molecules,cofactors, peptide aptamers, peptidoglycan, lipopolysaccharide, smallmolecules, endotoxins (e.g., bacterial lipopolysaccharides), and anycombinations thereof.

In some embodiments, the capture probe and/or label probe comprisesmicrobe-binding agents or molecules. In some embodiments, thetarget-binding molecules comprise microbe-binding molecules. Anymolecule or material that can bind to a microbe can be employed as themicrobe-binding molecule. Exemplary microbe-binding molecules (ormicrobe-binding molecules) include, but are not limited to, opsonins,lectins, antibodies and antigen binding fragments thereof, proteins,peptides, nucleic acids, carbohydrates, lipids, and any combinationsthereof. The microbe-binding molecule can comprise at least one (e.g.,one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty or more) microbe surface-binding domain (“microbebinding domain”). The term “microbe surface-binding domain” as usedherein refers to any molecules or a fragment thereof that canspecifically bind to the surface of a microbe, e.g., any componentpresent on a surface of a microbe.

Materials or substances which can serve as microbe-binding moleculesinclude, for example, peptides, polypeptides, proteins, peptidomimetics,antibodies, antibody fragments (e.g., antigen binding fragments ofantibodies), carbohydrate-binding protein, e.g., a lectin,glycoproteins, glycoprotein-binding molecules, amino acids,carbohydrates (including mono-, di-, tri- and poly-saccharides), lipids,steroids, hormones, lipid-binding molecules, cofactors, nucleosides,nucleotides, nucleic acids (e.g., DNA or RNA, analogues and derivativesof nucleic acids, or aptamers), peptidoglycan, lipopolysaccharide, smallmolecules, and any combinations thereof. The microbe-binding moleculecan be covalently (e.g., cross-linked) or non-covalently linked to thesubstrate surface.

In some embodiments, the microbe surface-binding domain can comprise anopsonin or a fragment thereof. The term “opsonin” as used herein refersto naturally-occurring and synthetic molecules which are capable ofbinding to or attaching to the surface of a microbe or a pathogen, ofacting as binding enhancers for a process of phagocytosis. Examples ofopsonins which can be used in the engineered molecules described hereininclude, but are not limited to, vitronectin, fibronectin, complementcomponents such as C1q (including any of its component polypeptidechains A, B and C), complement fragments such as C3d, C3b and C4b,mannose-binding protein, conglutinin, surfactant proteins A and D,C-reactive protein (CRP), alpha2-macroglobulin, and immunoglobulins, forexample, the Fc portion of an immunoglobulin.

In some embodiments wherein the target analyte comprises a microbe or afragment thereof, the capture probe and/or label probe can comprise acarbohydrate recognition domain derived from a carbohydrate-bindingmolecule. Examples of a carbohydrate-binding molecule include, but arenot limited to, lectin, collectin, ficolin, mannose-binding lectin(MBL), maltose-binding protein, arabinose-binding protein,glucose-binding protein, Galanthus nivalis agglutinin, peanut lectin,lentil lectin, DC-SIGN, C-reactive protein, and any combinationsthereof. In some embodiments, the label probe comprises a carbohydraterecognition domain and a reporter enzyme. In some embodiments, thecapture probe or label probe is a fusion peptide comprising acarbohydrate recognition domain of a lectin. In a label probe, thefusion peptide is conjugated to a reporter enzyme. For example, thefusion peptide can be a FcMBL, which is a fusion peptide comprisingmannan-binding lectin and a Fc portion of an immunoglobulin, and isdescribed in the U.S. application Ser. No. 13/574,191 entitled“Engineered Opsonin for Pathogen Detection and Treatment” and U.S.application Ser. No. 14/233,553 entitled “Engineered Microbe-TargetingMolecules and Uses Thereof,” both of which the patent applications areincorporated herein by reference. In some embodiments, a label probe canbe a FcMBL conjugated to an enzyme label (e.g., but not limited to,horseradish peroxidase, alkaline phosphatase, a glucose oxidase,tyrosinase, urease, a DNAzyme, a aptazyme, etc.). Label probes such asFcMBL-HRP or FcMBL-AP described in U.S. application Ser. No. 14/233,553entitled “Engineered Microbe-Targeting Molecules and Uses Thereof,”incorporated by reference, can be also used herein.

In some embodiments, the microbe surface-binding domain comprises acarbohydrate recognition domain or a carbohydrate recognition portionthereof. As used herein, the term “carbohydrate recognition domain”refers to a region, at least a portion of which, can bind tocarbohydrates on a surface of a microbe (e.g., a pathogen).

In some embodiments, the microbe surface-binding domain comprises alectin or a carbohydrate recognition or binding fragment or portionthereof. The term “lectin” as used herein refers to any moleculesincluding proteins, natural or genetically modified, that interactspecifically with saccharides (i.e., carbohydrates). The term “lectin”as used herein can also refer to lectins derived from any species,including, but not limited to, plants, animals, insects andmicroorganisms, having a desired carbohydrate binding specificity.Examples of plant lectins include, but are not limited to, theLeguminosae lectin family, such as ConA, soybean agglutinin, peanutlectin, lentil lectin, and Galanthus nivalis agglutinin (GNA) from theGalanthus (snowdrop) plant. Other examples of plant lectins are theGramineae and Solanaceae families of lectins. Examples of animal lectinsinclude, but are not limited to, any known lectin of the major groupsS-type lectins, C-type lectins, P-type lectins, and I-type lectins, andgalectins. In some embodiments, the carbohydrate recognition domain canbe derived from a C-type lectin, or a fragment thereof. C-type lectincan include any carbohydrate-binding protein that requires calcium forbinding. In some embodiments, the C-type lectin can include, but are notlimited to, collectin, DC-SIGN, and fragments thereof. Without wishingto be bound by theory, DC-SIGN can generally bind various microbes byrecognizing high-mannose-containing glycoproteins on their envelopesand/or function as a receptor for several viruses such as HIV andHepatitis C.

In some embodiments, the microbe-binding molecules or microbe-bindingmolecules can comprise a microbe-binding portion of the C-type lectins,including, e.g., but not limited to, soluble factors such as Collectins(e.g., MBL, surfactant protein A, surfactant protein D and Collectin11), ficolins (e.g. L-Ficolin, Ficolin A), receptor based lectins (e.g.,DC-SIGN, DC-SIGNR, SIGNR1, Macrophage Mannose Receptor 1, Dectin-1 andDectin-2), lectins from the shrimp Marsupenaeus japonicus (e.g. LectinA, Lectin B and Lectin C), or any combinations thereof.

In some embodiments, the microbe-binding molecules can comprise at leasta portion of non-C-type lectins (e.g., but not limited to, Wheat GermAgglutinin).

In some embodiments, the microbe-binding molecules can comprise at leasta portion of lipopolysaccharide (LPS)-binding proteins and/or endotoxinbinding proteins (e.g., but not limited to, CD14, MD2,lipopolysaccharide binding proteins (LBP), limulus anti-LPS factor(LAL-F), or any combinations thereof).

In some embodiments, the microbe-binding molecules can comprise at leasta portion of peptidoglycan binding proteins (e.g., but not limited to,mammalian peptidoglycan recognition protein-1 (PGRP-1), PGRP-2, PGRP-3,PGRP-4, or any combinations thereof.

Collectins are soluble pattern recognition receptors (PRRs) belonging tothe superfamily of collagen containing C-type lectins. Exemplarycollectins include, without limitations, mannan-binding lectin (MBL) ormannose-binding protein, surfactant protein A (SP-A), surfactant proteinD (SP-D), collectin liver 1 (CL-L1), collectin placenta 1 (CL-P1),conglutinin, collectin of 43 kDa (CL-43), collectin of 46 kDa (CL-46),and a fragment thereof.

In some embodiments, the microbe-surface binding domain comprises thefull amino acid sequence of a carbohydrate-binding protein. In someembodiments, the microbe-surface binding domain comprises a sequence ofa carbohydrate recognition domain of a carbohydrate-binding protein.Examples of carbohydrate-binding proteins include, but are not limitedto, lectin, collectin, ficolin, mannose-binding lectin (MBL),maltose-binding protein, arabinose-binding protein, glucose-bindingprotein, Galanthus nivalis agglutinin, peanut lectin, lentil lectin,DC-SIGN, C-reactive protein (CRP), and any combinations thereof.

In some embodiments, the microbe surface-binding molecule comprises amannose-binding lectin (MBL) or a carbohydrate binding fragment orportion thereof. Mannose-binding lectin, also called mannose bindingprotein (MBP), is a calcium-dependent serum protein that can play a rolein the innate immune response by binding to carbohydrates on the surfaceof a wide range of microbes or pathogens (viruses, bacteria, fungi,protozoa) where it can activate the complement system. MBL can alsoserve as a direct opsonin and mediate binding and uptake of microbes orpathogens by tagging the surface of a microbe or pathogen to facilitaterecognition and ingestion by phagocytes. MBL and an engineered form ofMBL (FcMBL and Akt-FcMBL) are described in the International ApplicationPublication Nos. WO/2011/090954 (corresponding U.S. patent applicationSer. No. 13/574,191 entitled “Engineered opsonin for pathogen detectionand treatment”) and WO/2013/012924 (corresponding U.S. patentapplication Ser. No. 14/233,553 entitled “Engineered microbe-targetingmolecules and uses thereof”), contents of both of which are incorporatedherein by reference.

In some embodiments, the microbe surface-binding molecule comprises atleast a portion of C-reactive protein that binds to a microbe orfragment thereof. Microbe-binding molecules comprising a portion ofC-reactive protein described in U.S. Provisional App. No. 61/917,705entitled “CRP Capture/Detection of Gram Positive Bacteria,” the contentsof which are incorporated herein by reference.

Without wishing to be bound by a theory, microbe binding moleculescomprising lectins or modified versions thereof can act asbroad-spectrum microbe binding molecules (e.g., pathogen bindingmolecules). Accordingly, antibiotic susceptibility method utilizinglectins (e.g., MBL and genetically engineered version of MBL (FcMBL andAkt-FcMBL)) as broad-spectrum microbe binding molecules (e.g., pathogenbinding molecules) to capture and grow the microbes, can be carried outwithout identifying the microbe (e.g., pathogen), either for extractionor for antibiotic sensitivity testing.

In some embodiments, at least two microbe surface-binding domains (e.g.two, three, four, five, six, seven or more) microbe surface-bindingdomains, can be linked together to form a multimeric microbesurface-binding domain. In such embodiments, the distances betweenmicrobe surface-binding domains can be engineered to match with thedistance between the binding sites on the target microbe surface. Insome embodiments, the microbe surface-binding domain can be present in aform of a monomer, dimer, trimer, tetramer, pentamer, hexamer, or anentity comprising more than six sub-units.

A multimeric microbe surface-binding domain can have each of theindividual microbe surface-binding domains to be identical.Alternatively, a multimeric microbe surface-binding domain can have atleast one, at least two, or at least three microbe surface-bindingdomains different from the rest. In such embodiments, microbesurface-binding domains that share a common binding specificity formolecule on a microbe surface can be used. By way of example only, thefibrinogen-like domain of several lectins has a similar function to theCRD of C-type lectins including MBL, and function as pattern-recognitionreceptors to discriminate microbes or pathogens from self. One of suchlectins comprising the fibrinogen-like domain is serum ficolins.

Serum ficolins have a common binding specificity for GlcNAc(N-acetyl-glucosamine), elastin or GalNAc (N-acetyl-galactosamine). Thefibrinogen-like domain is responsible for the carbohydrate binding. Inhuman serum, two types of ficolin, known as L-ficolin (also called P35,ficolin L, ficolin 2 or hucolin) and H-ficolin (also called Hakataantigen, ficolin 3 or thermolabile b2-macroglycoprotein), have beenidentified, and both of them have lectin activity. L-ficolin recognizesGlcNAc and H-ficolin recognizes GalNAc. Another ficolin known asM-ficolin (also called P3 5-related protein, ficolin 1 or ficolin A) isnot considered to be a serum protein and is found in leucocytes and inthe lungs. L-ficolin and H-ficolin activate the lectin-complementpathway in association with MASPs. M-Ficolin, L-ficolin and H-ficolinhave calcium-independent lectin activity. Accordingly, in someembodiments, a microbe-binding molecule can comprise MBL and L-ficolincarbohydrate recognition domains, MBL and H-ficolin carbohydraterecognition domains, or a combination thereof.

Any art-recognized recombinant carbohydrate-binding proteins orcarbohydrate recognition domains can also be used in the microbe-bindingmolecules. For example, recombinant mannose-binding lectins, e.g., butnot limited to, the ones disclosed in the U.S. Pat. Nos. 5,270,199;6,846,649; and U.S. Patent App. Publication No. US 2004/0229212,contents of all of which are incorporated herein by reference, can beused in constructing a microbe-binding molecule.

The microbe binding molecule can further comprise at least one (e.g.,one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty or more) substrate surface binding domain (“substratebinding domain”) adapted for orienting the microbe binding domain awayfrom the substrate surface. As used herein, the term “substrate-bindingdomain” refers to any molecule that facilitates the conjugation of theengineered molecules described herein to a solid substrate or afunctionalized substrate. The microbe binding domain and the substratebinding domains can be linked by a linker. Similarly, the substratebinding domain and the substrate surface can be linked by a linker.

The substrate-binding domain can comprise at least one amino group thatcan non-covalently or covalently couple with functional groups on thesurface of the substrate (e.g. an analyte-specific electrode, ananoparticle, etc). For example, the primary amines of the amino acidresidues (e.g., lysine or cysteine residues) at the N-terminus or inclose proximity to the N-terminus of the microbe surface-binding domainscan be used to couple with functional groups on the substrate surface.

In some embodiments, the substrate-binding domain can comprise at leastone, at least two, at least three or more oligopeptides. The length ofthe oligonucleotide can vary from about 2 amino acid residues to about10 amino acid residues, or about 2 amino acid residues to about 5 aminoacid residues. Determination of an appropriate amino acid sequence ofthe oligonucleotide for binding with different substrates is well withinone of skill in the art. For example, an oligopeptide comprising anamino acid sequence of Alanine-Lysine-Threonine (AKT), which provides asingle biotinylation site for subsequent binding to streptavidin-coatedsubstrate. Such single biotinylation site can also enable the microbesurface binding domain of a microbe binding molecule to orient away fromthe substrate, and thus become more accessible to microbes or pathogens.See, for example, Witus et al. (2010) J. Am. Chem. Soc. 132(47):16812-17.

The microbe-binding molecules can contain sequences from the samespecies or from different species. For example, an interspecies hybridmicrobe-binding molecule can contain a linker, e.g., a peptide linker,from a murine species, and a human sequence from a carbohydraterecognition domain protein, provided that they do not provideunacceptable levels of deleterious effects. The engineeredmicrobe-binding molecules described herein can also include those thatare made entirely from murine-derived sequences or fully human.

General methods of preparing such microbe-binding molecules are wellknown in the art (Ashkenazi, A. and S. M. Chamow (1997), “Immunoadhesinsas research tools and therapeutic agents,” Curr. Opin. Immunol. 9(2):195-200, Chamow, S. M. and A. Ashkenazi (1996). “Immunoadhesins:principles and applications,” Trends Biotechnol. 14(2):52-60). In oneexample, an engineered microbe-binding molecule can be made by cloninginto an expression vector such as Fc-X vector as discussed in Lo et al.(1998) Protein Eng. 11:495 and PCT application no. PCT/US2011/021603,filed Jan. 19, 2011, contents of both of which is incorporated herein byreference.

In some embodiments, the microbe-binding molecule is a fusion protein orpeptide comprising (a) a carbohydrate recognition domain derived from acarbohydrate binding protein, and (b) a linker as defined herein. Insome embodiments, the fusion protein or peptide further comprise asubstrate binding domain at one of its terminus (e.g., N-terminus),which permits a microbe-binding molecule to attach to a solid substratesuch that the carbohydrate recognition domain points away from the solidsubstrate surface.

In one embodiment, the microbe-binding molecule comprises an MBL, acarbohydrate recognition domain of an MBL, or a genetically engineeredversion of MBL (FcMBL) as described in the International ApplicationPublication Nos. WO/2011/090954 (corresponding U.S. patent applicationSer. No. 13/574,191 entitled “Engineered opsonin for pathogen detectionand treatment”) and WO/2013/012924 (corresponding U.S. patentapplication Ser. No. 14/233,553 entitled “Engineered microbe-targetingmolecules and uses thereof”), contents of both of which are incorporatedherein by reference. Amino acid sequences for MBL and engineered MBLare:

(i) MBL full length (SEQ ID NO. 1):MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSSPGINGFPGKD GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPGNPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMARIKKWLTFSL GKQVGNKFFL TNGEIMTFEK VKALCVKFQASVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTGNRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPI (ii)MBL without the signal sequence (SEQ ID NO. 2):ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKGEPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKSPDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI (iii) Truncated MBL (SEQ ID NO. 3):AASERKALQT EMARIKKWLT FSLGKQVGNK FFLTNGEIMTFEKVKALCVK FQASVATPRN AAENGAIQNL IKEEAFLGITDEKTEGQFVD LTGNRLTYTN WNEGEPNNAG SDEDCVLLLK NGQWNDVPCS TSHLAVCEFP I (iv)Carbohydrate recognition domain (CRD) of MBL (SEQ ID NO. 4):VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGAIQNLIKEEAF LGITDEKTEG QFVDLTGNRL TYTNWNEGEPNNAGSDEDCV LLLKNGQWND VPCSTSHLAV CEFPI (v)Neck + Carbohydrate recognition domain of MBL (SEQ ID NO. 5):PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI (vi) FcMBL.81 (SEQ ID NO. 6):EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSPGAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI (vii) Akt-FcMBL (SEQ ID NO. 7):AKTEPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKF FLTNGEIMTFEKVKALCVKF QASVATPRNA AENGAIQNLI KEEAFLGITDEKTEGQFVDL TGNRLTYTNW NEGEPNNAGS DEDCVLLLKN GQWNDVPCST SHLAVCEFPI (viii)FcMBL.111 (SEQ ID NO. 8): EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GATSKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI

In some embodiments, microbe-binding molecule comprises an amino acidsequence selected from SEQ ID NO. 1-SEQ ID NO. 8.

In some embodiments, the label probe is conjugated to at least onereporter enzyme, as described herein. A label probe and a reporterenzyme can be linked to each other by a linker. In some embodiments, thelinker between the label probe and the reporter enzyme is an amide bond.In some embodiments, the linker between the label probe and the enzymeis a disulfide (S—S) bond.

As used herein, the term “linker” generally refers to a molecular entitythat can directly or indirectly connect two parts of a composition,e.g., at least one target-binding molecule and at least onesubstrate-binding domain or at least one enzyme and at least onetarget-binding molecule. In some embodiments, the linker can directly orindirectly connect to one or more target-binding molecules ortarget-binding domains.

Linkers can be configured according to a specific need, e.g., based onat least one of the following characteristics. By way of example only,in some embodiments, linkers can be configured to have a sufficientlength and flexibility such that it can allow for a target analytesurface-binding domain to orient accordingly with respect to at leastone carbohydrate on a microbe surface. In some embodiments, linkers canbe configured to allow multimerization of at least two engineeredtarget-binding molecules (e.g., to from a di-, tri-, tetra-, penta-, orhigher multimeric complex) while retaining biological activity (e.g.,microbe-binding activity). In some embodiments, linkers can beconfigured to facilitate expression and purification of the engineeredtarget- or microbe-binding molecule described herein. In someembodiments, linkers can be configured to provide at least onerecognition-site for proteases or nucleases. In addition, linkers shouldbe non-reactive with the functional components of the engineeredmolecule described herein (e.g., minimal hydrophobic or chargedcharacter to react with the functional protein domains such as a microbesurface-binding domain or a substrate-binding domain).

In some embodiments, a linker can be configured to have any length in aform of a peptide, a protein, or any combinations thereof. In someembodiments, the peptide linker can vary from about 1 to about 1000amino acids long, from about 10 to about 500 amino acids long, fromabout 30 to about 300 amino acids long, or from about 50 to about 150amino acids long. Longer or shorter linker sequences can be also usedfor the engineered target- or microbe-binding molecules describedherein. In one embodiment, the peptide linker has an amino acid sequenceof about 200 to 300 amino acids in length.

In some embodiments, a peptide linker can be configured to have asequence comprising at least one of the amino acids selected from thegroup consisting of glycine (Gly), serine (Ser), asparagine (Asn),threonine (Thr), methionine (Met) or alanine (Ala), or at least one ofcodon sequences encoding the aforementioned amino acids (i.e., Gly, Ser,Asn, Thr, Met or Ala). Such amino acids and corresponding nucleic acidsequences are generally used to provide flexibility of a linker.However, in some embodiments, other uncharged polar amino acids (e.g.,Gln, Cys or Tyr), nonpolar amino acids (e.g., Val, Leu, Ile, Pro, Phe,and Trp), or nucleic acid sequences encoding the amino acids thereof canalso be included in a linker sequence. In alternative embodiments, polaramino acids can be added to modulate the flexibility of a linker. One ofskill in the art can control flexibility of a linker by varying thetypes and numbers of residues in the linker. See, e.g., Perham, 30Biochem. 8501 (1991); Wriggers et al., 80 Biopolymers 736 (2005).

In alternative embodiments, a linker can be a chemical linker of anylength. In some embodiments, chemical linkers can comprise a direct bondor an atom such as oxygen or sulfur, a unit such as NH, C(O), C(O)NH,SO, SO₂, SO₂NH, or a chain of atoms, such as substituted orunsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl,substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstitutedC6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substitutedor unsubstituted C5-C12 heterocyclyl, substituted or unsubstitutedC3-C12 cycloalkyl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, NH, or C(O). In some embodiments, thechemical linker can be a polymer chain (branched or linear).

In some embodiments where the linker is a peptide, such peptide linkercan comprise at least a portion of an immunoglobulin, e.g., IgA, IgD,IgE, IgG and IgM including their subclasses (e.g., IgG1), or a modifiedthereof. In some embodiments, the peptide linker can comprise a portionof fragment crystallization (Fc) region of an immunoglobulin or amodified thereof. In such embodiments, the portion of the Fc region thatcan be used as a linker can comprise at least one region selected fromthe group consisting of a hinge region, a CH₂ region, a CH₃ region, andany combinations thereof. By way of example, in some embodiments, a CH₂region can be excluded from the portion of the Fc region as a linker. Inone embodiment, Fc linker comprises a hinge region, a CH₂ domain and aCH₃ domain. Such Fc linker can be used to facilitate expression andpurification of the engineered microbe-binding molecules describedherein. The N terminal Fc has been shown to improve expression levels,protein folding and secretion of the fusion partner. In addition, the Fchas a staphylococcal protein A binding site, which can be used forone-step purification protein A affinity chromatography. See Lo K M etal. (1998) Protein Eng. 11: 495-500. Further, such Fc linker have amolecule weight above a renal threshold of about 45 kDa, thus reducingthe possibility of engineered microbe-binding molecules being removed byglomerular filtration. Additionally, the Fc linker can allowdimerization of two engineered microbe-binding molecules to form adimer, e.g., a dimeric MBL molecule.

In various embodiments, the N-terminus or the C-terminus of the linker,e.g., the portion of the Fc region, can be modified. By way of exampleonly, the N-terminus or the C-terminus of the linker can be extended byat least one additional linker described herein, e.g., to providefurther flexibility, or to attach additional molecules. In someembodiments, the N-terminus of the linker can be linked directly orindirectly (via an additional linker) with a substrate-binding domainadapted for orienting the carbohydrate recognition domain away from thesubstrate. Exemplary Fc linked MBL (FcMBL and Akt-FcMBL) are describedin PCT application no. PCT/US2011/021603, filed Jan. 19, 2011, contentof which is incorporated herein by reference.

In some embodiments, the linker can be embodied as part of the microbesurface-binding domain.

In some embodiments, the distance between the microbe surface-bindingdomain and the substrate surface can range from about 50 angstroms toabout 5000 angstroms, from about 100 angstroms to about 2500 angstroms,or from about 200 angstroms to about 1000 angstroms.

In some embodiments, the linkers can be branched. For branched linkers,the linker can link together at least one (e.g., one, two, three, four,five, six, seven, eight, nine, ten or more) surface binding domain andat least one (e.g., one, two, three, four, five, six, seven, eight,nine, ten or more) microbe surface-binding domain.

When the label probe is a peptide, polypeptide or a protein, the enzymecan be linked at the N-terminus, the C-terminus, or at an internalposition of the microbe-binding molecule. Similarly, the enzyme can belinked by its N-terminus, C-terminus, or an internal position.

In some embodiments, the capture probe can be affixed to a solidsubstrate described herein to form a target-binding substrate.Non-limiting examples of a solid substrate include, but are not limitedto, an electrode (e.g. an analyte-specific electrode), a nanoparticle, ananotube, sensor, a protein scaffold, a lipid scaffold, a dendrimer,microparticle or a microbead, a microtiter plate, a medical apparatus orimplant, a microchip, and any combinations thereof.

The solid substrate can be made of any material, including, but notlimited to, metal, metal alloy, polymer, plastic, paper, glass, fabric,packaging material, biological material such as cells, tissues,hydrogels, proteins, peptides, nucleic acids, and any combinationsthereof.

Additional material that can be used to fabricate or coat a solidsubstrate include, without limitations, polydimethylsiloxane, polyimide,polyethylene terephthalate, polymethylmethacrylate, polyurethane,polyvinylchloride, polystyrene polysulfone, polycarbonate,polymethylpentene, polypropylene, polyvinylidine fluoride, polysilicon,polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene,polyacrylonitrile, polybutadiene, poly(butylene terephthalate),poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol),styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinylbutyral, polyvinylidenedifluoride, poly(vinyl pyrrolidone), and anycombination thereof.

A solid substrate surface can be functionalized or activated forconjugation with capture probes by any methods known in the art.Exemplary conjugations include, but are not limited to, a linker asdescribed herein, a covalent bond, amide bond, additions tocarbon-carbon multiple bonds, azide alkyne Huisgen cycloaddition,Diels-Alder reaction, disulfide linkage, ester bond, Michael additions,silane bond, urethane, nucleophilic ring opening reactions: epoxides,non-aldol carbonyl chemistry, cycloaddition reactions: 1,3-dipolarcycloaddition, temperature sensitive, radiation (IR, near-IR, UV)sensitive bond or conjugation agent, pH-sensitive bond or conjugationagent, non-covalent bonds (e.g., ionic charge complex formation,hydrogen bonding, pi-pi interactions, cyclodextrin/adamantly host guestinteraction) and the like. In some embodiments, a solid substratesurface can be functionalized with addition of silane coupling agents(e.g., but not limited to organosilanes, aminosilanes, vinyl silanes,methacryl silanes, and any combinations thereof).

In some embodiments, the target-binding agents can comprisetarget-binding nanoparticles. As used herein, the term “target-bindingnanoparticles” refers to nanoparticles conjugated to capture probes forspecific binding with a target analyte.

In some embodiments, the target-binding nanoparticles can be magnetic(e.g., paramagnetic, superparamagnetic, or ferromagnetic). For example,the target-binding nanoparticles can be paramagnetic orsuperparamagnetic. The target-binding nanoparticles can range in sizefrom 1 nm to 1000 nm. For example, the target-binding nanoparticles canbe about 2.5 nm to about 500 nm, or about 5 nm to about 250 nm in size.In some embodiments, the target-binding nanoparticles can be about 5 nmto about 100 nm in size. In some embodiments, the target-bindingnanoparticles can be about 0.01 nm to about 10 nm in size. In someembodiments, the target-binding nanoparticles can be about 0.05 nm toabout 5 nm in size. In some embodiments, the target-bindingnanoparticles can be about 80 nm to about 1000 nm in size. In someembodiments, the target-binding nanoparticles can have a size rangingfrom about 1 nm to about 1000 nm, from about 10 nm to about 500 nm, fromabout 25 nm to about 300 nm, from about 40 nm to about 250 nm, or fromabout 50 nm to about 200 nm. In one embodiment, the target-bindingnanoparticles can have a size of about 50 nm to about 200 nm. Thetarget-binding magnetic nanoparticles can be manipulated using magneticfield or magnetic field gradient. Such particles commonly consist ofmagnetic elements such as iron, nickel and cobalt and their oxidecompounds. Magnetic microbeads are well-known and methods for theirpreparation have been described in the art. See, e.g., U.S. Pat. Nos.6,878,445; 5,543,158; 5,578,325; 6,676,729; 6,045,925; and 7,462,446;and U.S. Patent Publications No. 2005/0025971; No. 2005/0200438; No.2005/0201941; No. 2005/0271745; No. 2006/0228551; No. 2006/0233712; No.2007/01666232; and No. 2007/0264199, the contents of which areincorporated herein by reference.

The target-binding nanoparticles can be of any shape, including but notlimited to spherical, rod, elliptical, cylindrical, and disc. Thenanoparticles can be of any shape, e.g., a sphere. In some embodiments,the term “nanoparticle” as used herein can encompass a nanosphere. Theterm “nanosphere” as used herein refers to a nanoparticle having asubstantially spherical form. A substantially spherical nanoparticle isa nanoparticle with a difference between the smallest radii and thelargest radii generally not greater than about 40% of the smaller radii,and more typically less than about 30%, or less than 20%.

The capture probe or label probe can be present in any form, includingbut not limited to a target-binding molecule, and/or a target-bindingsubstrate (e.g., a target-binding molecule conjugated to a solidsubstrate) as described above. By “target-binding molecules” is meantherein molecules that can interact with or bind to a target analyte suchthat the target analyte can be captured or detected from a fluid sample.Typically the nature of the interaction or binding is noncovalent, e.g.,by hydrogen, electrostatic, or van der Waals interactions, however,binding can also be covalent. Target-binding molecules can benaturally-occurring, recombinant or synthetic. Examples of thetarget-binding molecule can include, but are not limited to an antibodyor a portion thereof, an antibody-like molecule, an enzyme, an antigen,a small molecule, a protein, a peptide, a peptidomimetic, acarbohydrate, an aptamer, and any combinations thereof. By way ofexample only, in immunohistochemistry, the target-binding molecule canbe an antibody specific to the target antigen to be analyzed. Anordinary artisan can readily identify appropriate target-bindingmolecules for each target species or analytes of interest to be detectedin various bioassays.

In some embodiments, the target-binding molecules can be modified by anymeans known to one of ordinary skill in the art. Methods to modify eachtype of target-binding molecules are well recognized in the art.Depending on the types of target-binding molecules, an exemplarymodification includes, but is not limited to genetic modification,biotinylation, labeling (for detection purposes), chemical modification(e.g., to produce derivatives or fragments of the target-bindingmolecule), and any combinations thereof. In some embodiments, thetarget-binding molecule can be genetically modified. In someembodiments, the target-binding molecule can be biotinylated. In someembodiments, the label probe is functionalized with biotin and at leastone reporter enzyme is conjugated to streptavidin. After the targetanalyte is bound to the capture probes on the analyte-specificelectrode, the target analyte complex is labeled with the biotinylatedlabel probe. Then, the streptavidin conjugated to at least one reporterenzyme binds to the biotin-functionalized label probe. In someembodiments, streptavidin may be conjugated with 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more reporter enzymes. Reporter enzymes that can be conjugatedto streptavidin include, but are not limited to, horseradish peroxidase(HRP), alkaline phosphatase (AP), glucose oxidase (GOx), tyrosinase,urease, a DNAzyme, an aptazyme, or any combinations thereof.

In some embodiments, the target-binding molecules can comprise on theirsurfaces microbe-binding molecules as described herein, and/or disclosedin WO/2011/090954 (corresponding U.S. patent application Ser. No.13/574,191 entitled “Engineered opsonin for pathogen detection andtreatment”) and WO/2013/012924 (corresponding U.S. patent applicationSer. No. 14/233,553 entitled “Engineered microbe-targeting molecules anduses thereof”), the contents of which are incorporated herein byreference. Accordingly, in some embodiments, the method described hereincan be used with the target-binding nanoparticles for microbial capture,i.e., microbe-binding nanoparticles, e.g., but not limited toFcMBL-coated nanoparticles. In some embodiments, the nanoparticles aremagnetic. Examples of microbe-binding magnetic particles can include,but are not limited to the ones described in WO/2011/090954(corresponding U.S. patent application Ser. No. 13/574,191 entitled“Engineered opsonin for pathogen detection and treatment”) andWO/2013/012924 (corresponding U.S. patent application Ser. No.14/233,553 entitled “Engineered microbe-targeting molecules and usesthereof”), the contents of which are incorporated herein by reference.

In some embodiments, the target-binding molecule can be an antibody or aportion thereof, or an antibody-like molecule. In some embodiments, thetarget-binding molecule can be an antibody or a portion thereof, or anantibody-like molecule that is specific for detection of a rare-cell,e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe biomarker. In some embodiments, the target-binding molecule canbe an antibody or a portion thereof, or an antibody-like molecule thatis specific for a protein or an antigen present on the surface of a rarecell, e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe. In such embodiments, the target-binding molecules can be usedto, for example, detect and/or identify cell type or species (includingnormal and/or diseased cells), the presence of cell or disease markers,cellular protein expression levels, phosphorylation or otherpost-translation modification state, or any combinations thereof.

In some embodiments, the target-binding molecule can be a protein or apeptide. In some embodiments, the protein or peptide can be essentiallyany proteins that can bind to a rare cell, e.g., a circulating tumorcell, a fetal cell, a stem cell and/or a microbe. By way of exampleonly, if the target species is a bacteria, exemplary proteins orpeptides that can be used to generate microbe-binding molecules and/ormicrobe-binding magnetic particles can include, but are not limited to,innate-immune proteins (e.g., without limitations, MBL, Dectin-1, TLR2,and TLR4 and any molecules (including recombinant or engineered proteinmolecules) disclosed here as well as the microbe-binding moleculesdisclosed in the International Application Publication Nos.WO/2011/090954 and WO/2013/012924, the content of which is incorporatedherein by reference) and proteins comprising the chitin-binding domain,and any factions thereof. Such innate-immune proteins and chitin-bindingdomain proteins can be used to detect their correspondingpattern-recognition targets (e.g., microbes such as bacteria) andfungus, respectively.

In some embodiments, the target-binding molecule can be an aptamer. Insome embodiments, the target-binding molecule can be a DNA or RNAaptamer. The aptamers can be used in various bioassays, e.g., in thesame way as antibodies or nucleic acids described herein. For example,the DNA or RNA aptamer can encode a nucleic acid sequence correspondingto a rare cell biomarker or a fraction thereof, for use as atarget-binding molecule on the nanoparticles described herein.

In some embodiments, the target-binding molecule can be a cell surfacereceptor ligand. As used herein, a “cell surface receptor ligand” refersto a molecule that can bind to the outer surface of a cell. Exemplarycell surface receptor ligand includes, for example, a cell surfacereceptor binding peptide, a cell surface receptor binding glycopeptide,a cell surface receptor binding protein, a cell surface receptor bindingglycoprotein, a cell surface receptor binding organic compound, and acell surface receptor binding drug. Additional cell surface receptorligands include, but are not limited to, cytokines, growth factors,hormones, antibodies, and angiogenic factors. In some embodiments, anyart-recognized cell surface receptor ligand that can bind to a rarecell, e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe, can be used as a target-binding molecule on the magneticparticles described herein.

In accordance with various embodiments described herein, a sample,including any fluid or specimen (processed or unprocessed) that isintended to be evaluated for the presence of a target analyte can besubjected to methods, compositions, kits and systems described herein.The sample or fluid can be liquid, supercritical fluid, solutions,suspensions, gases, gels, slurries, and combinations thereof. The sampleor fluid can be aqueous or non-aqueous.

In some embodiments, the sample can be an aqueous fluid. As used herein,the term “aqueous fluid” refers to any flowable water-containingmaterial that is suspected of comprising a pathogen.

In some embodiments, the sample can include a biological fluid obtainedfrom a subject. Exemplary biological fluids obtained from a subject caninclude, but are not limited to, blood (including whole blood, plasma,cord blood and serum), lactation products (e.g., milk), amniotic fluids,sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate,perspiration, mucus, liquefied stool sample, synovial fluid, lymphaticfluid, tears, tracheal aspirate, and any mixtures thereof. In someembodiments, a biological fluid can include a homogenate of a tissuespecimen (e.g., biopsy) from a subject. In one embodiment, a test samplecan comprises a suspension obtained from homogenization of a solidsample obtained from a solid organ or a fragment thereof.

In some embodiments, the sample can be a whole blood sample obtainedfrom a subject suspected of having a microbe infection (e.g., a pathogeninfection).

In some embodiments, the sample can include a fluid or specimen obtainedfrom an environmental source. For example, the fluid or specimenobtained from the environmental source can be obtained or derived fromfood products or industrial food products, food produce, poultry, meat,fish, beverages, dairy products, water (including wastewater), surfaces,ponds, rivers, reservoirs, swimming pools, soils, food processing and/orpackaging plants, agricultural places, hydrocultures (includinghydroponic food farms), pharmaceutical manufacturing plants, animalcolony facilities, and any combinations thereof.

In some embodiments, the sample can include a fluid or specimencollected or derived from a biological culture. For example, abiological culture can be a cell culture. Examples of a fluid orspecimen collected or derived from a biological culture includes the oneobtained from culturing or fermentation, for example, of single- ormulti-cell organisms, including prokaryotes (e.g., bacteria) andeukaryotes (e.g., animal cells, plant cells, yeasts, fungi), andincluding fractions thereof. In some embodiments, the test sample caninclude a fluid from a blood culture. In some embodiments, the culturemedium can be obtained from any source, e.g., without limitations,research laboratories, pharmaceutical manufacturing plants,hydrocultures (e.g., hydroponic food farms), diagnostic testingfacilities, clinical settings, and any combinations thereof.

In some embodiments, the sample can be a fluid or specimen collected orderived from a microbe colony.

In some embodiments, the sample can include a media or reagent solutionused in a laboratory or clinical setting, such as for biomedical andmolecular biology applications. As used herein, the term “media” refersto a medium for maintaining a tissue, an organism, or a cell population,or refers to a medium for culturing a tissue, an organism, or a cellpopulation, which contains nutrients that maintain viability of thetissue, organism, or cell population, and support proliferation andgrowth.

As used herein, the term “reagent” refers to any solution used in alaboratory or clinical setting for biomedical and molecular biologyapplications. Reagents include, but are not limited to, salinesolutions, PBS solutions, buffered solutions, such as phosphate buffers,EDTA, Tris solutions, and any combinations thereof. Reagent solutionscan be used to create other reagent solutions. For example, Trissolutions and EDTA solutions are combined in specific ratios to create“TE” reagents for use in molecular biology applications.

In some embodiments, the sample can be a non-biological fluid. As usedherein, the term “non-biological fluid” refers to any fluid that is nota biological fluid as the term is defined herein. Exemplarynon-biological fluids include, but are not limited to, water, saltwater, brine, buffered solutions, saline solutions, sugar solutions,carbohydrate solutions, lipid solutions, nucleic acid solutions,hydrocarbons (e.g. liquid hydrocarbons), acids, gasolines, petroleum,liquefied samples (e.g., liquefied samples), and mixtures thereof.

Advantageously, the methods described herein are useful for detectingvery low amounts and/or concentrations of one or more target analytes ina sample. In some embodiments, the method comprises mixing a samplecomprising the target analyte(s) with a plurality of nanoparticles,wherein the nanoparticles are functionalized as described herein withone or more types of capture probes, each type of capture probe beingspecific for binding with one target analyte, and allowing the targetanalyte(s) to bind with the capture probes on the nanoparticles. Bindingthe target analyte to nanoparticles prior to detection increases thesensitivity of the assay. Then, the sample comprising the target analytebound to nanoparticles is introduced into an electrochemical sensorcomprising a fluid-contact surface and one or more analyte-specificelectrodes immobilized on at least a portion of the fluid-contactsurface, wherein the analyte-specific electrode is functionalized with acapture probe for specific binding with the target analyte. Next, thetarget analyte bound to nanoparticles is allowed to bind with thecapture probe on the analyte-specific electrode, thereby forming acomplex comprising nanoparticle, target analyte and capture probe on asurface of the analyte-specific electrode. Next, the complex is labeledwith a label probe, wherein the label probe binds specifically with thetarget analyte and the label probe is conjugated with at least onereporter enzyme. Next, an electroactive mediator precipitatingcomposition is introduced into the electrochemical sensor, wherein areaction of the electroactive mediator precipitating composition withthe at least one reporter enzyme conjugated with the label probe formsan electroactive precipitate locally adsorbed at the surface of theanalyte-specific electrode. Then a voltage is applied to theelectrochemical sensor, wherein the voltage corresponds to the standardredox potential of the electroactive precipitate, and a currentgenerated from the analyte-specific electrode of the electrochemicalsensor is measured to detect the target analyte.

The methods described herein can be used to diagnose an illness in apatient. Non-limiting examples include detection of a microbe in a bloodsample, which can be used to diagnose a patient with an infection.Another non-limiting example includes detection of abnormal cells in ablood sample (e.g., cancer cells). The methods described herein can alsobe used to detect microbes in food, water, the environment, etc.

In the methods described herein, it can be necessary or desired that asample is preprocessed prior to being introduced into an electrochemicalsensor as described herein, e.g., with a preprocessing reagent. Even incases where pretreatment is not necessary, preprocessing optionally canbe done for mere convenience (e.g., as part of a regimen on a commercialplatform). A preprocessing reagent can be any reagent appropriate foruse with the methods described herein.

The sample preprocessing step generally comprises adding one or morereagents to the sample. This preprocessing can serve a number ofdifferent purposes, including, but not limited to, hemolyzing cells suchas blood cells, dilution of sample, etc. The preprocessing reagents canbe present in the sample container before sample is added to the samplecontainer or the preprocessing reagents can be added to a sample alreadypresent in the sample container. When the sample is a biological fluid,the sample container can be a VACUTAINER®, e.g., a heparinizedVACUTAINER®.

The preprocessing reagents include, but are not limited to, surfactantsand detergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, lipase, collagenase,cellulases, amylases and the like), and solvents, such as buffersolutions.

In some embodiments, a preprocessing reagent is a surfactant or adetergent. In one embodiment, the preprocessing reagent is Triton X100.

After addition of the preprocessing reagent, the reagent can be mixedinto the sample. This can be simply accomplished by agitating thesample, e.g., shaking the sample and/or moving the sample around in amicrofluidic device.

After the optional preprocessing step, the sample can be optionallyfurther processed by adding one or more processing reagents to thesample. These processing reagents can degrade unwanted molecules presentin the sample and/or dilute the sample for further processing. Theseprocessing reagents include, but are not limited to, surfactants anddetergents, salts, cell lysing reagents, anticoagulants, degradativeenzymes (e.g., proteases, lipases, nucleases, lipase, collagenase,cellulases, amylases, heparanases, and the like), and solvents, such asbuffer solutions. Amount of the processing reagent to be added candepend on the particular sample to be analyzed, the time required forthe sample analysis, identity of the microbe to be detected or theamount of microbe present in the sample to be analyzed.

It is not necessary, but if one or more reagents are to be added theycan present in a mixture (e.g., in a solution, “processing buffer”) inthe appropriate concentrations. Amount of the various components of theprocessing buffer can vary depending upon the sample, microbe to bedetected, concentration of the microbe in the sample, or time limitationfor analysis.

Generally, addition of the processing buffer can increase the volume ofthe sample by 5%, 10%, 15%, 20% or more. In some embodiments, about 50μl to about 500 μl of the processing buffer are added for each ml of thesample. In some embodiments, about 100 μl to about 250 μl of theprocessing buffer are added for each ml of the sample. In oneembodiment, about 125 μl of the processing buffer are added for each mlof the sample.

In some embodiments, a detergent or surfactant comprises about 5% toabout 20% of the processing buffer volume. In some embodiments, adetergent or surfactant comprises about 5% to about 15% of theprocessing buffer volume. In one embodiment, a detergent or surfactantcomprises about 10% of the processing buffer volume.

In some embodiments, one mL of the processing buffer comprises about 1 Uto about 100 U of a degradative enzyme. In some embodiments, one ml ofthe processing buffer comprises about 5 U to about 50 U of a degradativeenzyme. In one embodiment, one ml of the processing buffer comprisesabout 10U of a degradative enzyme. Enzyme unit (U) is an art known termfor the amount of a particular enzyme that catalyzes the conversion of 1μmol of substrate per minute.

In some embodiments, one ml of the processing buffer comprises about 1μg to about 10 μg, or about lmg to about 10 mg of an anti-coagulant. Insome embodiments, one ml of the processing buffer comprises about 1 μgto about 5 μg, or about lmg to about 5 mg of an anti-coagulant. In oneembodiment, one ml of the processing buffer comprises about 4.6 μg, orabout 4.6 mg of an anti-coagulant.

Exemplary anti-coagulants include, but are not limited to, heparin,heparin substitutes, salicylic acid, D-phenylalanyl-L-prolyl-L-argininechloromethyl ketone (PPACK), Hirudin, ANCROD® (snake venom, VIPRONAX®),tissue plasminogen activator (tPA), urokinase, streptokinase, plasmin,prothrombopenic anticoagulants, platelet phosphodiesterase inhibitors,dextrans, thrombin antagonists/inhibitors, ethylene diamine tetraaceticacid (EDTA), acid citrate dextrose (ACD), sodium citrate, citratephosphate dextrose (CPD), sodium fluoride, sodium oxalate, sodiumpolyanethol sulfonate (SPS), potassium oxalate, lithium oxalate, sodiumiodoacetate, lithium iodoacetate and mixtures thereof.

Generally, salt concentration of the processing buffer can range fromabout 10 mM to about 100 mM. In some embodiments, the processing buffercomprises a salt at a concentration of about 25 mM to about 75 mM. Insome embodiment, the processing buffer comprises a salt at aconcentration of about 45 mM to about 55 mM. In one embodiment, theprocessing buffer comprises a salt at a concentration of about 43 mM toabout 45 mM.

The processing buffer can be made in any suitable buffer solution knownto a skilled artisan. In some embodiments, the buffer solution isphysiologically compatible to cells. Alternatively, the processingbuffer can be made in water.

In some embodiments, the processing buffer comprises a mixture ofTriton-X, DNAse I, human plasmin, CaC12 and Tween-20. In one embodiment,the processing buffer consists of a mixture of Triton-X, DNAse I, humanplasmin, CaC12 and Tween-20 in a TBS buffer.

In one embodiment, one ml of the processing buffer comprises 100 μl ofTriton-X100, 10 μl of DNAse (1 U/1 l), 10 μl of human plasmin at 4.6mg/ml and 870 μl of a mixture of TBS, 0.1% Tween-20 and 50 mM CaCl₂.

After processing of the sample, the sample can be subjected to a targetanalyte capture process. The target analyte capture process can allowfor concentrating and/or cleaning up the sample before proceeding withdetection. The extraction and concentration process can be completed inless than 6 hours, less than 5 hours, less than 4 hours, less than 3hours, less than 2 hours, less than 1 hour, less than 30 minutes, lessthan 15 minutes, less than 10 minutes, or shorter. In some embodiments,extraction and concentration of a target analyte in the sample can bedone within 10 minutes to 60 minutes of starting the process. In someembodiments, extraction and concentration of a target analyte in thesample can be done in about 10 minutes, e.g., mixing a sample comprisinga target analyte to be extracted with at least one target-bindingsubstrate (e.g., a plurality of target-binding magnetic nanoparticlesdescribed herein) optionally followed by separation of the target-boundtarget-binding substrate from the rest of the sample.

Additionally, the extraction and concentration process described hereincan be utilized to extract a target analyte in a sample of any givenvolume. In some embodiments, sample volume is about 0.25 ml to about 50ml, about 0.5 ml to about 25 ml, about 1 ml to about 15 ml, about 2 mlto about 10 ml. In some embodiments, sample volume is about 5 ml. In oneembodiment, sample volume is 8 ml.

In some embodiments, the target analyte capture process comprises mixinga solid substrate, the surface of which is coated with target-bindingmolecules which can bind to a target analyte in the sample. By “coated”is meant that a layer of target-binding molecules is present on asurface of the solid substrate and available for binding with a microbe.A solid substrate or a solid supporting structure coated withtarget-binding molecules is also referred to as a “target-bindingsubstrate.” For example, an analyte-specific electrode as describedherein can be a target-binding substrate. The amount of thetarget-binding molecules used to coat a solid substrate surface can varywith a number of factors such as a solid substrate surface area, coatingdensity, types of target-binding molecules, and binding performance. Askilled artisan can determine the optimum density of target-bindingmolecules on a solid substrate surface using any methods known in theart. By way of example only, the amount of the target-binding moleculesused to coat a solid substrate can vary from about 1 wt % to about 30 wt% or from about 5 wt % to about 20 wt %. In some embodiments, the amountof the target-binding molecules used to coat the solid substrate can behigher or lower, depending on a specific need. However, it should benoted that if the amount of the target-binding molecules used to coatthe substrate is too low, the target-binding substrate can show a lowerbinding performance with a target analyte. On the contrary, if theamount of the target-binding molecules used to coat the substrate is toohigh, the dense layer of the target-binding molecules can exert anadverse influence on the binding properties.

In some embodiments, the target-binding substrate is a particle, e.g., anano- or micro-particle. In some embodiments, the target-bindingmolecule coated substrate is a MBL, a recombinant MBL, FcMBL orAKT-FcMBL coated bead, microbead or magnetic microbead as described inthe International Application Publication Nos. WO/2011/090954(corresponding U.S. patent application Ser. No. 13/574,191 entitled“Engineered opsonin for pathogen detection and treatment”) andWO/2013/012924 (corresponding U.S. patent application Ser. No.14/233,553 entitled “Engineered microbe-targeting molecules and usesthereof”), contents of both of which are incorporated herein byreference. In some embodiments, the target-binding substrate can becoated with antibodies, aptamers, or nucleic acids against specificmicrobes, lectin (e.g., but not limited to MBL), or any combinationsthereof.

After addition of the target-binding substrate, the target-bindingsubstrate can be mixed in the sample to allow target analytes to bindwith the capture probe. This can be simply accomplished by agitating thesample, e.g., shaking the sample and/or moving the sample around in amicrofluidic device.

The sample mixture may optionally be subjected to a target analyteseparation process. Without wishing to be bound by a theory, in someembodiments, capture and separation of the bound target analytes fromthe sample can concentrate the target analytes. In some embodiments,capture and separation of the bound target analytes from the sample candeplete target analytes from a sample. In some embodiments, capture andseparation of the bound target analytes from the sample can removecomponents which can interfere with the assay from the bound targetanalytes. Any method known in the art for separating the target-bindingsubstrate from the sample can be employed.

For example, when the target-binding substrate is magnetic, e.g., amagnetic bead, a magnet can be employed to separate the substrate boundtarget analytes from the sample fluid. Without limitations, targetanalyte capture also can be carried out by non-magnetic means, forexample, by coating microbe-binding molecules on non-magnetic solidsubstrates or scaffolds (e.g., beads, posts, fibers, filters, capillarytubes, etc.) and flow sample by these affinity substrates.

The skilled artisan is well aware of methods for carrying out magneticseparations. Generally, a magnetic field or magnetic field gradient canbe applied to direct the magnetic beads. Optionally, the bound targetanalyte can be washed with a buffer to remove any leftover sample andunbound components. Without wishing to be bound by a theory, capture andseparation of the bound target analytes from the sample can concentratethe target analytes and also remove components, which can interfere withthe assay or process, from the test sample. In some embodiments, themagnetic field gradient can be generated by a magnetic field gradientgenerator described in the U.S. Provisional Application No. 61/772,360,entitled “Magnetic Separator.”

In some embodiments, the capture or detection of a microbe from thebiological fluid or other samples can be accomplished by a method thatdoes not require the identity of the microbe to be known for capture ordetection. This can be accomplished using a solid substrate coated witha broad-spectrum microbe-binding molecule for microbe extraction fromthe sample. For example, in their previous work, the inventors describeda method for the extraction and concentration of microbes (e.g.,pathogens) from blood that does not require prior identification ofpathogen. See PCT Application No. PCT/US2011/021603, filed Jan. 19,2011, content of which is incorporated herein by reference. The methodis based on beads that are coated with mannose binding lectin (MBL) or agenetically engineered version of MBL (FcMBL or Akt-FcMBL). MBL is a keycomponent of the innate immune system, which binds to carbohydratestructures containing mannose, N-acetyl glucosamine and fucose on thesurface of microbes or pathogens and that are not found on mammaliancells. MBL binds to at least 36 species of bacteria (e.g. Gram positive:Staphylococci, MRSA, VRSA, Streptococci, Clostridium; Gram negative:Pseudomonas, E. coli, Klebsiella,), 17 viruses (e.g. CMV, HIV, Ebola,HSV, HepB), 20 fungi (e.g., Candida, Aspergillus, Cryptococcus), and 9parasites (e.g. Malaria, Schistosoma), in addition to at least onemolecular toxin (e.g., LPS endotoxin). Consequently, MBL can serve as abroad-spectrum capture reagent, allowing a wide range of microbes (e.g.,pathogens) to be extracted and concentrated from blood samples or otherbiological fluids.

Accordingly, in some embodiments of the aspects described herein,microbe capture or detection from a biological sample or other sample isby substrate coated with a broad-spectrum microbe-binding molecule. Forexample, microbe capture or extraction from a biological sample is bymagnetic micro- or nano-beads as described in the InternationalApplication Publication Nos. WO/2011/090954 (corresponding U.S. patentapplication Ser. No. 13/574,191 entitled “Engineered opsonin forpathogen detection and treatment”) and WO/2013/012924 (correspondingU.S. patent application Ser. No. 14/233,553 entitled “Engineeredmicrobe-targeting molecules and uses thereof”), contents of both ofwhich are incorporated herein by reference.

A sample comprising at least one target analyte, optionally pre-treatedor pre-mixed with capture probe functionalized nanoparticles, can beintroduced into an electrochemical sensor to detect and/or analyze thepresence of the target analyte(s). In some embodiments, theelectrochemical sensor comprises at least one analyte-specific electrodeon a fluid-contact surface therein, wherein the analyte-specificelectrode is functionalized with a capture probe for specific bindingwith the target analyte. The target analytes are allowed to bind withthe capture probe on the analyte-specific electrode, thereby forming acomplex comprising the target analyte and the capture probe on a surfaceof the analyte-specific electrode. In some embodiments, label probesthat can bind with the target analytes can then be used to label thetarget analytes for detection. As used herein, a “label probe” refers toa molecule that comprises a reporter enzyme and can bind with a targetanalyte. Label probes can include, but are not limited to, MBL or aportion thereof, FcMBL, AKT-FcMBL, wheat germ agglutinin, lectins,antibodies (e.g., gram-negative antibodies or gram-positive antibodies,antibiotics to specific microbial strains or species), antigen bindingfragments of antibodies, aptamers, carbohydrate-binding proteins,peptides, polypeptides, cell-binding molecules, lipid-binding molecules,ligands (agonists or antagonists) of cell-surface receptors and thelike.

In some embodiments, the label probe can comprise MBL or atarget-binding molecule described herein. In one embodiment, the labelprobe comprises FcMBL. Without wishing to be bound by a theory, labelprobes based on MBL, and FcMBL in particular, attach selectively to abroad range of microbes, and so they enable the method described hereinto detect the majority of blood-borne microbes with high sensitivity andspecificity.

In some embodiments, the reporter enzyme comprises horseradishperoxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOx),tyrosinase, urease, a DNAzyme, a aptazyme, or any combination thereof.

In one embodiment, the label probe can comprise a MBL or a portionthereof, or a FcMBL molecule linked to a HRP. Conjugation of HRP to anyproteins and antibodies are known in the art. In one embodiment,FcMBL-HRP construct is generated by direct coupling HRP to FcMBL usingany commercially-available HRP conjugation kit. In some embodiments, thetarget analytes bound on an analyte-specific electrode can be incubatedwith the HRP-labeled target-binding molecules, e.g., MBL or a portionthereof, or a FcMBL molecule linked to a HRP for a period of time, e.g.,at least about 5 mins, at least about 10 mins, at least about 15 mins,at least about 20 mins, at least about 25 mins, at least about 30 mins.The typical concentrations of HRP-labeled molecules used in the assaycan range from about 1:500 to about 1:20,000 dilutions. In oneembodiment, the concentration of HRP-labeled label probes, e.g., MBL ora portion thereof, or a FcMBL molecule linked to a HRP molecule, can beabout 1:1000 to about 1:10000 dilutions.

In one embodiment, the label probe can comprise a MBL or a portionthereof, or a FcMBL molecule linked to an AP. Conjugation of AP to anyproteins and antibodies are known in the art. In one embodiment,FcMBL-AP construct is generated by direct coupling AP to FcMBL using anycommercially-available AP conjugation kit. In some embodiments, thetarget analytes bound on an analyte-specific electrode can be incubatedwith the AP-labeled target-binding molecule, e.g., MBL or a portionthereof, or a FcMBL molecule linked to a AP for a period of time, e.g.,at least about 5 mins, at least about 10 mins, at least about 15 mins,at least about 20 mins, at least about 25 mins, at least about 30 mins.The typical concentrations of AP-labeled molecules used in the assay canrange from about 1:1000 to about 1:20,000 dilutions. In one embodiment,the concentration of AP-labeled target-binding molecules, e.g., MBL or aportion thereof, or a FcMBL molecule linked to a AP molecule, can beabout 1:5000 to about 1:10000 dilutions.

Following incubation with the label probe, the analyte-specificelectrodes may optionally be washed with a wash buffer one or more(e.g., 1, 2, 3, 4, 5 or more) times to remove any unbound probes. Insome embodiments, the wash buffer used after incubation with a labelprobe can contain calcium ions at a concentration of about at leastabout 0.01 mM, at least about 0.05 mM, at least about 0.1 mM, at leastabout 0.5 mM, at least about 1 mM, at least about 2.5 mM, at least about5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM,at least about 40 mM, at least about 50 mM or more. In alternativeembodiments, the wash buffer used after incubation with a label probecan contain no calcium ions. In some embodiments, the wash buffer usedafter incubation with a label probe can contain a chelating agent. Awash buffer can be any art-recognized buffer used for washing betweenincubations with antibodies and/or labeling molecules. An exemplary washbuffer can include, but is not limited to, phosphate-buffered saline(PBS), Tris-buffered saline (TBS), TBST, a mixture of TBS, 0.1% Tweenand 5 mM Ca2+, and any combination thereof.

The amount of calcium ions (Ca2+) present in the processing bufferand/or wash buffer can vary from about 1 mM to about 100 mM, from about3 mM to about 50 mM, or from about 5 mM to about 25 mM. Calcium ions canbe obtained from any calcium salts, e.g., but not limited to, CaCl₂,CaBr₂, CaI₂, and Ca(NO₃)₂, and any other art-recognized calcium salts.Without wishing to be bound by theory, the presence of calcium ions inthe processing buffer and/or wash buffer can facilitate and/or maintaincalcium-dependent binding (e.g., lectin-mediated binding such asMBL-mediated binding) of the microbe to a microbe-binding substrate.

In some embodiments, the wash buffer can exclude calcium ions and/orinclude a chelator, e.g., but not limited to, EDTA. In such embodiments,microbes that solely depend on calcium-dependent binding (e.g.,lectin-mediated binding such as MBL-mediated binding) to themicrobe-binding substrate will less likely bind to the microbe-bindingsubstrate in the absence of calcium ions. However, microbes (e.g.,pathogens such as S. aureus) that at least partly depend onnon-calcium-dependent interaction (e.g., but not limited to, proteinA/Fc-mediated binding) with the microbe-binding substrate (e.g.,FcMBL-coated magnetic particles) can bind to the microbe-bindingsubstrate in the absence of calcium ions, and additional information canbe found, e.g., in the International Application Publication No.WO/2013/012924, or in the U.S. Provisional App. No. 61/605,052 filedFeb. 29, 2012, the content of which is incorporated herein by reference.

In some embodiments, without wishing to be bound by theory, it can bedesirable to use a wash buffer without a surfactant or a detergent forthe last wash before addition of the electroactive mediatorprecipitating composition, because a surfactant or detergent may haveadverse effect to the enzymatic reaction with the enzyme substrate andelectroactive mediator. The electrochemical sensor can optionally bewashed any number (e.g., 1, 2, 3, 4, 5 or more) of times beforedetection. Without wishing to be bound by a theory, such washing canreduce and/or eliminate any contaminants from the biological fluid thatcan be problematic during detection.

In some embodiments, an electroactive mediator precipitating compositioncan be added to develop the assay. After the electroactive mediatorcomposition reacts with reporter enzymes on the bound and labeled targetanalytes and forms an electroactive precipitate locally adsorbed at thesurface of the analyte-specific electrodes, the electrochemical sensormay be optionally washed with wash buffer to remove any electroactivemediator precipitating composition or electroactive precipitate that isnot adsorbed at the analyte-specific electrode surface. Any wash bufferas described herein may be used. An exemplary wash buffer can include,but is not limited to, phosphate-buffered saline (PBS), Tris-bufferedsaline (TBS), TBST, a mixture of TBS, 0.1% Tween and 5 mM Ca2+, and anycombination thereof.

To detect any target analyte bound to the analyte-specific electrodes, avoltage is applied to the electrochemical sensor, wherein the voltagecorresponds to the standard redox potential of the electroactiveprecipitate locally adsorbed at the surface of the analyte-specificelectrode. Then, a current generated from the analyte-specific electrodeof the electrochemical sensor is measured to detect the target analyte.Without being bound by theory, the voltage applied to theelectrochemical sensor corresponds to an electrochemical reduction oroxidation potential of the electroactive mediator in a fully orpartially oxidized state, and the generated current corresponds to areduction or oxidation current derived from reduction or oxidation ofthe oxidized electroactive mediator. In some embodiments, the voltageapplied is about −0.2V to +0.2V versus a reference electrode.

Any processes or steps described herein can be performed by a module ordevice. While these are discussed as discrete processes, one or more ofthe processes or steps described herein can be combined into one systemfor carrying out the assays of any aspects described herein. In someembodiments, the assay or process described herein can be adapted foruse in a high-throughput platform, e.g., an automated system orplatform.

In addition to the above mentioned components, any embodiments of thekits described herein can include informational material. Theinformational material can be descriptive, instructional, marketing orother material that relates to the methods described herein and/or theuse of the aggregates for the methods described herein. For example, theinformational material can describe methods for using the kits providedherein to perform an assay for capture and/or detection of a targetanalyte, e.g., a microbe. The kit can also include an empty containerand/or a delivery device, e.g., which can be used to deliver a testsample to a test container.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is a link or contact information,e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments, the kit can contain separate containers, dividersor compartments for each component and informational material. Forexample, each different component can be contained in a bottle, vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, acollection of magnetic nanoparticles is contained in a bottle, vial orsyringe that has attached thereto the informational material in the formof a label.

Exemplary embodiments of the inventon are also descrbied by one or moreof the following numbered paragrphs:

-   -   1. A method for detecting a target analyte in a sample,        comprising:        -   (a) introducing a sample comprising a target analyte into an            electrochemical sensor comprising a fluid-contact surface            and an analyte-specific electrode immobilized on at least a            portion of the fluid-contact surface, wherein the            analyte-specific electrode is functionalized with a capture            probe for specific binding with the target analyte;        -   (b) allowing the target analyte to bind with the capture            probe on the analyte-specific electrode, thereby forming a            complex comprising the target analyte and the capture probe            on a surface of the analyte-specific electrode;        -   (c) labeling the complex with a label probe, wherein the            label probe binds specifically with the target analyte and            the label probe is conjugated with at least one reporter            enzyme;        -   (d) introducing an electroactive mediator precipitating            composition into the electrochemical sensor, wherein a            reaction of the electroactive mediator precipitating            composition with the at least one reporter enzyme conjugated            with the label probe forms an electroactive precipitate            locally adsorbed at the surface of the analyte-specific            electrode;        -   (e) applying a voltage to the electrochemical sensor,            wherein the voltage corresponds to the standard redox            potential of the electroactive precipitate; and        -   (f) measuring a current generated from the analyte-specific            electrode of the electrochemical sensor to detect the target            analyte;    -    wherein the target analyte is not a nucleic acid.    -   2. The method of paragraph 1, further comprising prior to step        (a):    -    i. mixing a sample comprising the target analyte with a        plurality of nanoparticles, wherein at least one nanoparticle of        said plurality of nanoparticles is functionalized with a capture        probe for specific binding with the target analyte; and    -    ii. allowing the target analyte to bind with the capture probe        on said at least one nanoparticle.    -   3. The method of paragraph 1 or 2, wherein the electrochemical        sensor comprises a plurality of analyte-specific electrodes        immobilized on at least a portion of the fluid-contact surface,        wherein each analyte-specific electrode in said plurality of        analyte-specific electrodes is functionalized with a capture        probe for specific binding with a specific target analyte.    -   4. The method of paragraph 3, wherein at least two of the        analyte-specific electrodes are adapted to detect different        target analytes.    -   5. The method of any one of paragraphs 1-4, wherein at least two        different target analytes in the sample are detected.    -   6. The method of any one of paragraphs 1-5, wherein the target        analyte is selected from the group consisting of a protein, a        peptide, a polypeptide, a peptidomimetic, an antibody, an        antibody fragment, an amino acid, a peptide aptamer, a        peptidoglycan, a cell, microbial matter, a carbohydrate, an        antigen, a lipid, a steroid, a hormone, a lipopolysaccharide, an        endotoxin, a drug, a lipid-binding molecule, a cofactor, a small        molecule, a toxin, and any combination thereof.    -   7. The method of paragraph 6, wherein the protein is a        glycoprotein.    -   8. The method of paragraph 6, wherein the microbial matter is        selected from the group consisting of bacteria, viruses,        protozoa, fungi, yeast, microbes, parasites, any fragments        thereof, and any combination thereof.    -   9. The method of paragraph 6, wherein the carbohydrate is        selected from the group consisting of mannose, mannan, N-acetyl        glucosamine, fucose, a monosaccharide, a disaccharide, a        trisaccharide, a polysaccharide, and any combination thereof.    -   10. The method of any one of paragraphs 2-9 wherein the        nanoparticle is a magnetic nanoparticle, a gold nanoparticle, a        silver nanoparticle, a semiconductor nanoparticle, or a        polymeric nanoparticle.    -   11. The method of any one of paragraphs 2-10, wherein at least        two of the nanoparticles are functionalized with capture probes        for specific binding with at least two different target        analytes.    -   12. The method of any one of paragraphs 1-11, further        comprising, prior to the step of applying the voltage to the        electrochemical sensor, washing the electrochemical sensor to        remove any electroactive mediator precipitating composition or        electroactive precipitate that is not adsorbed at the        analyte-specific electrode surface.    -   13. The method of any one of paragraphs 1-12, wherein the        electrochemical sensor comprises one or more microfluidic flow        cells.    -   14. The method of any one of paragraphs 1-13, wherein the        electrochemical sensor comprises one or more open wells.    -   15. The method of any one of paragraphs 1-14, wherein the        analyte-specific electrode is a planar or 3-dimensional        electrode.    -   16. The method of any one of paragraphs 1-15, wherein the        analyte-specific electrode comprises gold, silver, copper,        platinum, aluminum, stainless steel, tungsten, indium tin oxide,        titanium, lead, nickel, palladium, silicon, polyimide, parylene,        benzocyclobutene, carbon, graphite, or any combination thereof.    -   17. The method of any one of paragraphs 1-16, wherein the        fluid-contact surface further comprises a counter electrode and        a reference electrode immobilized thereon.    -   18. The method of any one of paragraphs 1-17, wherein the        fluid-contact surface further comprises a positive control        electrode and/or a negative control electrode immobilized        thereon.    -   19. The method of any one of paragraphs 1-18, wherein the        voltage applied to the electrochemical sensor corresponds to an        electrochemical reduction or oxidation potential of the        electroactive mediator in a fully or partially oxidized state.    -   20. The method of any one of paragraphs 1-19, wherein the        generated current corresponds to a reduction or oxidation        current derived from reduction of the fully or partially        oxidized electroactive mediator.    -   21. The method of any one of paragraphs 1-20, wherein the        voltage window is about −0.2V to +0.2V versus a reference        electrode.    -   22. The method of any one of paragraphs 1-21, wherein the        fluid-contact surface is a non-electrically conductive surface.    -   23. The method of paragraph 22, wherein the non-electrically        conductive surface comprises plastic, poly(carbonate) (PC),        poly(methyl methacrylate) (PMMA), cyclic olefin polymers (COP),        cyclic olefin copolymers (COC), silicon nitride, parylene,        kapton, styrene-ethylene-butylene-styrene (SEBS),        poly-dimethysiloxane (PDMS), polyimide, silicon dioxide, and any        combination thereof.    -   24. The method of any one of paragraphs 1-23, wherein the        capture probe and the label probe are independently selected        from the group consisting of an antibody, an antibody fragment,        a carbohydrate-binding protein, a peptide, a polypeptide, an        aptamer, a cell-binding molecule, a lipid-binding molecule, a        polynucleotide, a lipid, a carbohydrate, and any combination        thereof.    -   25. The method of paragraph 24, wherein the target analyte        comprises a microbe, and the capture probe and label probe        comprise a carbohydrate binding protein, wherein the        carbohydrate binding protein comprises a carbohydrate        recognition domain of mannan-binding lectin (MBL).    -   26. The method of paragraph 25, wherein the carbohydrate        recognition domain of MBL is conjugated to an Fc portion of an        immunoglobin.    -   27. The method of any one of paragraphs 1-26, wherein said at        least one reporter enzyme is conjugated to the label probe        before the label probe binds to the target analyte complex.    -   28. The method of any one of paragraphs 1-26, wherein said at        least one reporter enzyme is conjugated to the label probe after        the label probe binds to the target analyte complex.    -   29. The method of any one of paragraphs 1-28, wherein the label        probe is functionalized with biotin and said at least one        reporter enzyme is conjugated to streptavidin.    -   30. The method of paragraph 29, wherein the label probe first        binds to the target analyte complex, and then the streptavidin        conjugated to said at least one reporter enzyme binds to the        biotin functionalized label probe.    -   31. The method of any one of paragraphs 1-30, wherein the at        least one reporter enzyme comprises horseradish peroxidase        (HRP), alkaline phosphatase (AP), glucose oxidase (GOx),        tyrosinase, urease, a DNAzyme, an aptazyme, or any combination        thereof.    -   32. The method of paragraph 31, wherein the at least one        reporter enzyme comprises HRP.    -   33. The method of any one of paragraphs 1-32, wherein the        electroactive mediator precipitating composition comprises a        reporter enzyme substrate and an electroactive mediator.    -   34. The method of paragraph 33, wherein the reporter enzyme        substrate is selected from the group consisting of hydrogen        peroxide, carbamide peroxide, nucleotides, oligonucleotides,        RNA, DNA, phosphorylated peptides, phosphorylated proteins,        phosphorylated small molecules, glucose, phenols, tyrosine,        dopamine, catechol, urea, and any combination thereof.    -   35. The method of paragraph 34, wherein the reporter enzyme        substrate is hydrogen peroxide.    -   36. The method of any one of paragraphs 33-35, wherein the        electroactive mediator is selected from the group consisting of        3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine        dihydrochloride (OPD), 2,2′-Azinobis        [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), p-Nitrophenyl        Phosphate (PNPP), 3,3′-diaminobenzidine (DAB),        4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate        (BCIP), nitro blue tetrazolium (NBT), methylene blue,        hydroquinone, ferrocene derivatives, and any combination        thereof.    -   37. The method of paragraph 36, wherein the electroactive        mediator is TMB.    -   38. The method of any one of paragraphs 1-37, wherein the        electroactive mediator precipitating composition further        comprises a precipitating agent.    -   39. The method of paragraph 38, wherein the precipitating agent        is selected from the group consisting of a water-soluble        polymer, a pyrrolidinone polymer, a polyaniline, a polypyrrole,        a polythiophene, alginic acid, methyl vinyl ether/maleic        anhydride copolymer, dextran sulfate, carrageenan, and any        combination thereof.    -   40. The method of paragraph 39, wherein the precipitating agent        is a pyrrolidinone polymer.    -   41. A kit for electrochemical multiplex detection of a plurality        of target analytes in a sample comprising:        -   (a) an electrochemical sensor comprising a fluid-contact            surface and a plurality of analyte-specific electrodes            immobilized on at least a portion of the fluid-contact            surface, wherein the analyte-specific electrodes are each            functionalized with a capture probe for binding a specific            target analyte;        -   (b) a plurality of label probes, wherein each label probe is            for binding a specific target analyte, and wherein each            label probe is conjugated to at least one reporter enzyme or            is functionalized to be conjugated to at least one reporter            enzyme; and        -   (c) an electroactive mediator precipitating composition            comprising a reporter enzyme substrate, an electroactive            mediator and a precipitating agent, wherein a reaction of            the reporter enzyme substrate and the electroactive mediator            with the reporter enzyme forms an electroactive precipitate            locally adsorbed at the surface of the analyte-specific            electrodes;    -    wherein none of the target analytes are nucleic acids.    -   42. The kit of paragraph 41, further comprising a plurality of        nanoparticles, wherein at least one nanoparticle of said        plurality of nanoparticles is functionalized with a capture        probe for specific binding with a target analyte.    -   43. The kit of paragraph 42, wherein the nanoparticles are        independently selected from a magnetic nanoparticle, a gold        nanoparticle, a silver nanoparticle, a semiconductor        nanoparticle, or a polymeric nanoparticle.    -   44. The kit of any one of paragraphs 41-43, wherein the        electrochemical sensor comprises one or more open wells.    -   45. The kit of any one of paragraphs 41-44, wherein the        electrochemical sensor comprises one or more microfluidic flow        cells.    -   46. The kit of any one of paragraphs 41-45, wherein the capture        probe and the label probe are independently selected from the        group consisting of an antibody, an antibody fragment, a        carbohydrate-binding protein, a peptide, a polypeptide, an        aptamer, a cell-binding molecule, a lipid-binding molecule, and        any combination thereof.    -   47. The kit of paragraph 46, wherein the target analyte        comprises a microbe, and the capture probe and label probe        comprise a carbohydrate binding protein, wherein the        carbohydrate binding protein comprises a carbohydrate        recognition domain of mannan-binding lectin (MBL).    -   48. The method of paragraph 47, wherein the carbohydrate        recognition domain of MBL is conjugated to an Fc portion of an        immunoglobin.    -   49. The kit of any one of paragraphs 41-48, wherein the label        probes are functionalized with biotin and the reporter enzymes        are conjugated to streptavidin, so that the label probes first        bind to specific target analytes, and then the streptavidin        conjugated to the reporter enzymes binds to the biotin        functionalized label probes.    -   50. The kit of any one of paragraphs 41-49, wherein the at least        one reporter enzyme comprises horseradish peroxidase (HRP),        alkaline phosphatase (AP), glucose oxidase (GOx), tyrosinase,        urease, a DNAzyme, a aptazyme, or any combination thereof.    -   51. The kit of paragraph 50, wherein the at least one reporter        enzyme comprises HRP.    -   52. The kit of any one of paragraphs 41-51, wherein the        electroactive mediator precipitating composition comprises a        reporter enzyme substrate and an electroactive mediator.    -   53. The kit of paragraph 52, wherein the reporter enzyme        substrate is selected from the group consisting of hydrogen        peroxide, carbamide peroxide, nucleotides, oligonucleotides,        RNA, DNA, phosphorylated peptides, phosphorylated proteins,        phosphorylated small molecules, glucose, phenols, tyrosine,        dopamine, catechol, urea, and any combination thereof.    -   54. The kit of paragraph 53, wherein the reporter enzyme        substrate is hydrogen peroxide.    -   55. The kit of any one of paragraphs 41-54, wherein the        electroactive mediator is selected from the group consisting of        3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine        dihydrochloride (OPD), 2,2′-Azinobis        [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS), p-Nitrophenyl        Phosphate (PNPP), 3,3′-diaminobenzidine (DAB),        4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate        (BCIP), nitro blue tetrazolium (NBT), methylene blue,        hydroquinone, ferrocene derivatives, and any combination        thereof.    -   56. The kit of paragraph 55, wherein the electroactive mediator        is TMB.    -   57. The kit of any one of paragraphs 41-56, wherein the        electroactive mediator precipitating composition further        comprises a precipitating agent.    -   58. The kit of paragraph 57, wherein the precipitating agent is        selected from the group consisting of a water-soluble polymer, a        pyrrolidinone polymer, a polyanaline, a polypyrrole, a        polythiophene, alginic acid, methyl vinyl ether/maleic anhydride        copolymer, dextran sulfate, carrageenan, and any combination        thereof.    -   59. The kit of paragraph 58, wherein the precipitating agent is        a pyrrolidinone polymer.    -   60. An electrochemical sensor comprising:        -   (a) a fluid-contact surface and a plurality of            analyte-specific electrodes immobilized on at least a            portion of the fluid-contact surface, wherein the            analyte-specific electrodes are each functionalized with a            capture probe for binding a specific target analyte;        -   (b) a plurality of different nanoparticle-bound target            analytes bound to the corresponding capture probes of the            analyte-specific electrodes; and        -   (c) an electroactive precipitate locally adsorbed at the            surfaces of at least some of the analyte-specific            electrodes, wherein the electroactive precipitate is formed            from a reaction of an electroactive mediator precipitating            composition comprising a reporter enzyme substrate, an            electroactive mediator and a precipitating agent, with a            reporter enzyme coupled to the nanoparticle-bound target            analytes;    -    wherein none of the target analytes are nucleic acids.    -   61. The electrochemical sensor of paragraph 60, wherein the        reporter enzyme is coupled to the nanoparticle-bound target        analytes by specific binding of a label probe to the        corresponding nanoparticle-bound target analytes, wherein the        label probe is conjugated to the reporter enzyme.

Some Selected Definitions

For convenience, certain terms employed in the entire application(including the specification, examples, and appended claims) arecollected here. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means ±1%.

In one aspect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising”). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

As used interchangeably herein, the terms “microbes” and “pathogens”generally refer to microorganisms, including bacteria, fungi, protozoan,archaea, protists, e.g., algae, and a combination thereof. The term“microbes” also includes pathogenic microbes, e.g., bacteria causingdiseases such as plague, tuberculosis and anthrax; protozoa causingdiseases such as malaria, sleeping sickness and toxoplasmosis; fungicausing diseases such as ringworm, candidiasis or histoplasmosis; andbacteria causing diseases such as sepsis. The term “microbe” or“microbes” can also encompass non-pathogenic microbes, e.g., somemicrobes used in industrial applications.

In some embodiments, the term “microbe” or “microbes” also encompassesfragments of microbes, e.g., cell components of microbes, LPS, and/orendotoxin.

As used herein, the term “binding” or “bound” generally refers to areversible binding of one agent or molecule to another agent or moleculevia, e.g., van der Waals force, hydrophobic force, hydrogen bonding,and/or electrostatic force. The binding interaction between an agent ormolecule and another agent or molecule can be described by adissociation constant (Ka) or association constant (K).

As used herein, the term “small molecules” refers to natural orsynthetic molecules including, but not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, aptamers, nucleotides, nucleotide analogs,organic or inorganic compounds (i.e., including heteroorganic andorganometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. These and other changes can be made to the disclosure inlight of the detailed description. All such modifications are intendedto be included within the scope of the appended claims.

Specific elements of any of the disclosed embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

EXAMPLES

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

Example 1

An electrode array consisting of 64 individually addressable 300 μm indiameter gold working electrodes sharing common reference and counterelectrodes was fabricated using standard microfabrication technology.Borofloat wafers were successively coated with the lift off resist LOR20and the imaging resist Shipley 2005. Following photopatterning of theelectrode array design (AutoCAD, Autodesk Inc, USA), the gold electrodearray pattern was revealed in devoloping solution CD30. Wafers were thensuccessively coated with 10 nm of titanium used as metal adhesion layer,and 80 nm of gold. The unwanted resist was lifts off in Remover PG at90° C. Finally, the electrode arrays were insulated with SU8-2002 andthe connection pads, working, counter and reference electrodes revealedphotolithographically before releasing individual electrode arrays usingan automated diamond saw.

The electrochemical characterization of the electrode array wasperformed using a PGSTAT12 potentiostat (Metrohm AG, The Netherlands)and an Ag/AgCl wire reference electrode and a platinum counterelectrode. All assays were realized using the on-chip reference andcounter electrodes and all electrodes were addressed simultaneouslyusing a dedicated 64-channel measuring system specifically developed.Simple microfluidic flow cells were fabricated using an Epilog Legend36EXT (EPIX) to cut and drill 2 mm thick poly(methylmethacrylate) sheetsand define the microfluidic channels in 100 μm thick medical gradedouble-sided adhesive tape (Adhesives Research Ltd., Ireland—ArCare90880).

Each of the assay electrodes was individually coated with FcMBL.Approximately 10 μL of diluted nanoparticle-bound target analyte wasinjected into the microfluidics and incubated for 60 minutes at roomtemperature. Channels were subsequently flushed with 100 μL of TBSbuffer before injecting 20 μL of HRP-labelled FcMBL label probe preparedat a concentration of 10 nM in buffer as well. Labelling of theelectrode-bound complexes occurred over a period of 30 minutes at roomtemperature, before flushing the microfluidics with 100 μL of TBSbuffer. The presence of HRP label was measured by fast chronoamperometryafter injecting 20 μL of TMB Enhanced HRP membrane substrate (DiarectAG, Germany), incubated for 5 minutes, and washing with 100 μL of Trisbuffer, before reading the array and measuring the reduction currentderived from the reduction of the HRP-oxidized TMB at −0.2 V (vs.internal reference).

Data were processed using a Visual Basic macro running under MS Excel totreat the current traces recorded at the 64 electrodes. The currentresponse at 500 ms was used as the signal. Limits of detection weretaken as the concentration value corresponding to the averaged currentresponse of the negative control sensors over the entire concentrationrange plus three times the averaged standard deviation.

Without being bound by theory, high background signal was found tooriginate from the active transport of the oxidised TMB (TMB_(ox))during its injection in the microfluidic cell. However, due to the highTMB:HRP reaction kinetics and the fluid dynamics, the TMB_(ox) generatedat one electrode can be actively transported to the next electrode evenminutes after the TMB injection, resulting in high background current.To limit the transport of TMB_(ox) to adjacent electrodes, aprecipitating TMB formulation was used. Precipitating TMB formulationsare commonly used in immunohistochemistry and Western blotting, andtypically contain additives such as alginic acid, methyl vinylether/maleic anhydride copolymer, dextran sulfate and/or carrageenan,which can readily precipitate TMB_(ox). The precipitated TMB was foundto conserve its electroactivity. More importantly, it formed a stableelectroactive precipitate at the electrode surface that could not bedissolved in aqueous buffer at pH 7.4. Consequently, following thecapture probe binding and HRP-labeling steps, the arrays were incubatedfor 5 minutes in precipitating TMB and flushed with 100 μL of Trisbuffer before reading the array.

Example 2

Dilute samples of rare target analytes (e.g., proteins, carbohydrates,pathogens, pathogen fragments, endotoxins, etc.) can be detectedelectrochemically, as shown in FIG. 1. Electrochemical sensors, i.e.electrodes (FIG. 1A), are modified with bio-engineered mannan-bindinglectin (FcMBL, FIG. 1B) capable of recognising mannan moieties presentat the surface of pathogen cell wall and endotoxins. Upon exposure to asample containing even low concentration of pathogens,pathogens/fragments/toxins will bind to the electrode surface (FIG. 1C).To quantify the amount bound, an enzyme (e.g. horseradish peroxidase(HRP)) modified FcMBL is introduced, which will further bind to thesurface captured pathogen (FIG. 1D). The amount of HRP present, which isproportional to the amount of captured pathogen, is finally quantifiedusing precipitating 3,3′,5,5′-tetramethylbenzidine (TMB). TMB in thepresence of HRP and hydrogen peroxide, will become oxidized (FIG. 1E),complexing with the precipitating agent and finally precipitating at theelectrode surface (FIG. 1F). Following a washing step, the precipitatedTMB is electrochemically detected. Detection sensitivity is increased bypreconcentrating the pathogen/fragment/toxins using nanoparticles coatedwith FcMBL in a first step. As illustrated in FIG. 1F, not allpathogen/pathogen fragment/toxin will bind to the electrode surface dueto mass transport limitations. Preconcentrating the pathogen/pathogenfragment/toxin in solution using nanoparticles and capturing thenanoparticle complexes at the electrode surface (FIG. 1H) will result inmore RhMBL-HRP binding to the electrode surface (FIG. 1I), a largeramount of TMB being oxidized and precipitating at the electrode surface(FIG. 1J), and consequently a higher detection signal.

Example 3

Pre-treating a sample with nanoparticles functionalized with captureprobes for specific binding with a target analyte increases sensorsensitivity, as shown in FIGS. 2A-2B. FIG. 2A (left) shows theelectrochemical detection of E. coli bacteria which were pre-treatedwith the antibiotic cefepime at a concentration of 100 μg/mL for 4 hoursat 37° C. FIG. 2B (right) shows the electrochemical detection ofuntreated E. coli. For both FIGS. 2A and 2B, E. coli was obtained asBioBall® (bioMerieux, 15 CFU/mL), and diluted in TBS-Tween Ca²⁺ bufferto 7.5 and 3.75 CFU/mL. The results presented in FIGS. 2A-2B wereobtained using a 64-electrode sensor array, each electrode being eithermodified with FcMBL or a monolayer of thiolated poly(ethylene glycol)used as negative control. Each data point is the average of threesensors. FIGS. 2A-2B demonstrate the sensitivity enhancement resultingfrom the preconcentration of treated and untreated E. coli usingFcMBL-coated nanoparticles. Particularly more pronounced at lowconcentrations, a 4-fold improvement in signal intensity was measuredwhen detecting 3.75 CFU/mL of antibiotic-treated E. coli.

Example 4

This example illustrates the difference in signal intensity betweenlabeling with single HRP vs. poly-HRP conjugated streptavidin, as shownin FIG. 5. The experiment was carried out in TBS buffer using a flowcell having analyte-specific electrodes functionalized with antibodiesraised against interleukin-6 (IL-6). IL-6 bound to the electrodes waslabeled with biotinylated anti-IL-6 antibodies, and then labeled withHRP-streptavidin or poly-HRP streptavidin. The flow cells were treatedwith H₂O₂/TMB precipitating composition, then washed with TBS buffer,and current readings were taken from the electrodes.Poly-HRP-streptavidin has 6-8 HRP per streptavidin label. Approximately3-fold sensitivity enhancement was obtained using poly-HRP streptavidin.IL-6 was detected at 2 pg/mL. Media: control. IL-6: 20 pg/mL. Mix: IL-8:IL-8: 5 ng/mL, GCSF: 125 pg/mL, Rantes: 125 pg/mL, IP10: 12.5 ng/mL. ,Mix+IL-6: Mix+IL-6 2 pg/mL.

Example 5

A full cross-reactivity study of anti-IL-6 modified electrochemicalsensors exposed to various individual chemokines and mixtures wasperformed, as shown in FIG. 6. It can be seen that the method and sensorare highly selective for IL-6 detection, and there is a lowfalse-positive reading for other chemokines. Media: control. IL-6: 20pg/mL. IL-8: 20 ng/mL. G-CSF: 500 pg/mL. Rantes: 500 pg/mL. IP10: 50ng/mL. Mix: IL-8: 5 ng/mL, GCSF: 125 pg/mL, Rantes: 125 pg/mL, IP10:12.5 ng/mL. Mix+IL-6: Mix+IL-6: 2 pg/mL.

Example 6

Electrochemical detection of the polysaccharide mannan was performed inTBS-Tween Ca²⁺ buffer using a biotin-FcMBL labeling approach, as shownin FIG. 7. Analyte-specific electrodes functionalized with FcMBL weretreated with various concentrations of mannan in TBS buffer. The boundmannan was labeled with biotinylated FcMBL and HRP-conjugatedstreptavidin. The electrodes were treated with H₂O₂/TMB precipitatingcomposition, then washed with TBS buffer, and current readings weretaken from the electrodes. This demonstrates the applicability of thesensor platform (also useful for protein detection) for detectingpolysaccharides present on bacteria cell walls.

Example 7

Electrochemical detection of the polysaccharide mannan was performed inTBS-Tween Ca²⁺ buffer using a HRP-RhMBL labeling approach, as shown inFIG. 8. Analyte-specific electrodes functionalized with FcMBL weretreated with various concentrations of mannan in TBS buffer. The boundmannan was labeled with HRP conjugated RhMBL. The electrodes weretreated with H₂O₂/TMB precipitating composition, then washed with TBSbuffer, and current readings were taken from the electrodes. Thisdemonstrates the applicability of the sensor platform (also useful forprotein detection) for detecting polysaccharides present on bacteriacell walls.

Example 8

A lung-on-a-chip was infected with rhinovirus. Following infection, thebasal and apical compartment of the chip was washed with PBS. Thecollected samples were analyzed using multiplex electrochemical assay.Calibration curves for the simultaneous electrochemical detection of thetwo inflammatory markers IP-10 and IL-6 in culture media are shown inFIGS. 12A and 12B. Standards containing both IP-10 and IL-6 at knownconcentration were prepared in culture media.

Evaluation of cross-reactivity and reproducibility data is shown inFIGS. 13A and 13B. One flow cell comprising sensors for IL-6 and sensorsfor IP-10, as well as negative control (i.e. No antibody attached) andpositive sensors (i.e. Biotin-modified) was filled with a samplecontaining IL-6 at a concentration of 100 pg/mL. The assay was completedby adding the biotin-labeled detection antibody, followed bystreptavidin-HRP and finally the precipitating TMB reagent. The IL-6specific sensors clearly indicated the presence of IL-6 in the samplewhile the signals measured at the IP-10 specific and the negativecontrol remained very low. The positive control sensor signal was veryhigh as expected, as it should bind large amount of streptavidin-HRP.Sensors sequentially modified with IL-6 or IP10 antibodies in a singleflow cell were exposed to either 250 pg/mL IL-6, or 250 mg/mL IP-10 or250 pg/mL of both protein. The responses measured indicate that no orlittle cross-reactivity occurs at the surface of the sensors.

Correlation of the electrochemical results and ELISA results ispresented in FIGS. 14A and 14B. Side-to-side comparison of thecalculated concentration measured using traditional ELISA kits (blackbars, 1 kit for each protein) and simultaneously measured on theelectrochemical plaftorm (light grey bars, NI: non infected chip; RV:Rhinovirus infected chip) shows that the correlation was excellent withan r² value of 0.9947. This experiment demonstrates multiplexedelectrochemical detection of several proteins in a single sample exitinga lung-on-a-chip infected with rhinovirus.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

What is claimed is:
 1. A method comprising: (a) introducingsimultaneously a reporter enzyme substrate, an electroactive mediatorand a precipitating agent into an electrochemical sensor, wherein theelectrochemical sensor comprises an electrode and a reporter enzymeconjugate, and wherein a reaction of the electroactive mediatorprecipitating composition with the reporter enzyme conjugate forms anelectroactive precipitate locally adsorbed at a surface of theelectrode; (b) applying a voltage to the electrochemical sensor, whereinthe voltage corresponds to the standard redox potential of theelectroactive precipitate; and (c) measuring a current generated fromthe electrode of the electrochemical sensor to detect a target analyte.2. The method of claim 1, wherein the electrochemical sensor comprises aplurality of electrodes.
 3. The method of claim 2, wherein at least twoelectrodes in the plurality of electrodes are adapted to detectdifferent target analytes.
 4. The method of claim 1, wherein theelectroactive mediator is selected from the group consisting of3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride(OPD), 2,2′-Azinobis [3 -ethylbenzothiazoline-6-sulfonic acid] (ABTS),p-Nitrophenyl Phosphate (PNPP), 3,3′-diaminobenzidine (DAB),4-chloro-1-naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP),nitro blue tetrazolium (NBT), methylene blue, hydroquinone, ferrocenederivatives, and any combination thereof.
 5. The method of claim 3,wherein the electroactive mediator is TMB.
 6. The method of claim 1,wherein the reporter enzyme substrate is selected from the groupconsisting, of hydrogen peroxide, carbamide peroxide, nucleotides,oligonucleotides, RNA, DNA, phosphorylated peptides, phosphorylatedproteins, phosphorylated small molecules, glucose, phenols, tyrosine,dopamine, catechol, urea, and any combination thereof.
 7. The method ofclaim 6, wherein the reporter enzyme substrate is hydrogen peroxide. 8.The method of claim 1, wherein the precipitating agent is selected fromthe group consisting of a water-soluble polymer, a pyrrolidinonepolymer, a polyaniline, a polypyrrole, a polythiophene, alginic acid,methyl vinyl ether/maleic anhydride copolymer, dextran sulfate,carrageenan, and any combination thereof.
 9. The method of claim 8,wherein the precipitating agent is a pyrrolidinone polymer.
 10. Themethod of claim 1, wherein the reporter enzyme conjugate comprises anenzyme conjugated with an antibody, an antibody fragment, acarbohydrate-binding protein, a peptide, a polypeptide, an aptamer, acell-binding molecule, a lipid-binding molecule, a polynucleotide, alipid, a carbohydrate, or any combinations thereof.
 11. The method ofclaim 1, wherein the reporter enzyme conjugate comprises an enzymeselected from the group consisting of horseradish peroxidase (HRP),alkaline phosphatase (AP), glucose oxidase (GOx), tyrosinase, urease, aDNAzyme, an aptazyme, and any combinations thereof.
 12. The method ofclaim 11, wherein the enzyme is HRP.
 13. The method of claim 1, whereinthe electrochemical sensor comprises one or more microfluidic flowcells.
 14. The method of claim 1, wherein the electrochemical sensorcomprises one or more open wells.
 15. The method of claim 1, wherein theelectrode is a planar or 3-dimensional electrode.
 16. The method ofclaim 1, wherein the electrochemical sensor further comprises a counterelectrode and a reference electrode.
 17. The method of claim 1, whereinthe electrochemical sensor further comprises a positive controlelectrode and/or a negative control electrode.
 18. The method of claim1, wherein the generated current corresponds to a reduction or oxidationcurrent derived from reduction of the fully or partially oxidizedelectroactive mediator.
 19. A kit for electrochemical multiplexdetection of a plurality of target analytes in a sample comprising: (a)an electrochemical sensor comprising a fluid-contact surface and aplurality of electrodes immobilized on at least a portion of thefluid-contact surface, wherein the electrodes are adapted to detectdifferent target analytes; (b) a plurality of reporter enzymeconjugates; and (c) an electroactive mediator precipitating compositioncomprising a reporter enzyme substrate, an electroactive mediator and aprecipitating agent, wherein a reaction of the reporter enzyme substrateand the electroactive mediator with a reporter enzyme conjugate in theplurality of reporter enzyme conjugates forms an electroactiveprecipitate locally adsorbed at a surface of at least one of theelectrodes.
 20. An electrochemical sensor comprising: (a) afluid-contact surface and a plurality of electrodes immobilized on atleast a portion of the fluid-contact surface, wherein the electrodes areadapted to detect different target analytes; (b) a plurality of reporterenzyme conjugates; and (c) an electroactive precipitate locally adsorbedat the surfaces of at least one of the electrodes, wherein theelectroactive precipitate is formed from a reaction of an electroactivemediator precipitating composition comprising a reporter enzymesubstrate, an electroactive mediator and a precipitating agent, with areporter enzyme conjugate in the plurality of reporter enzymeconjugates.